EP3632901B1 - Activateurs de l'histone acétyltransférase et leurs utilisations - Google Patents

Activateurs de l'histone acétyltransférase et leurs utilisations Download PDF

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EP3632901B1
EP3632901B1 EP19205945.9A EP19205945A EP3632901B1 EP 3632901 B1 EP3632901 B1 EP 3632901B1 EP 19205945 A EP19205945 A EP 19205945A EP 3632901 B1 EP3632901 B1 EP 3632901B1
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disease
compound
body weight
mice
hat
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EP3632901A1 (fr
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Yan Feng
Mauro Fa
Ottavio Arancio
Shixian Deng
Donald W. Landry
Yitshak Francis
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Columbia University in the City of New York
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/27—Esters, e.g. nitroglycerine, selenocyanates of carbamic or thiocarbamic acids, meprobamate, carbachol, neostigmine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/42—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/44—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring
    • C07C235/58—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring with carbon atoms of carboxamide groups and singly-bound oxygen atoms, bound in ortho-position to carbon atoms of the same non-condensed six-membered aromatic ring
    • C07C235/64—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring with carbon atoms of carboxamide groups and singly-bound oxygen atoms, bound in ortho-position to carbon atoms of the same non-condensed six-membered aromatic ring having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/39—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton at least one of the nitrogen atoms being part of any of the groups, X being a hetero atom, Y being any atom
    • C07C323/40—Y being a hydrogen or a carbon atom
    • C07C323/42—Y being a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/39—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton at least one of the nitrogen atoms being part of any of the groups, X being a hetero atom, Y being any atom
    • C07C323/43—Y being a hetero atom
    • C07C323/44—X or Y being nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/62—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C323/00—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups
    • C07C323/64—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and sulfur atoms, not being part of thio groups, bound to the same carbon skeleton
    • C07C323/67—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and sulfur atoms, not being part of thio groups, bound to the same carbon skeleton containing sulfur atoms of sulfonamide groups, bound to the carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/72—Nitrogen atoms
    • C07D213/75—Amino or imino radicals, acylated by carboxylic or carbonic acids, or by sulfur or nitrogen analogues thereof, e.g. carbamates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/81—Amides; Imides
    • C07D213/82—Amides; Imides in position 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D333/00—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom
    • C07D333/02—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings
    • C07D333/04—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom
    • C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D333/38—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals

Definitions

  • Cognitive neurodegenerative disorders are characterized by synaptic dysfunction, cognitive abnormalities, and/or the presence of inclusion bodies throughout the CNS containing, for example, but not limited to native beta-amyloid fragments, native and phosphorylated Tau, native and phosphorylated alpha-synuclein, lipofuscin, cleaved TARDBP (TDB-43), in various percentages and in relation to the specific disease.
  • native beta-amyloid fragments native and phosphorylated Tau
  • native and phosphorylated alpha-synuclein native and phosphorylated alpha-synuclein
  • lipofuscin cleaved TARDBP (TDB-43)
  • AD Alzheimer's disease
  • a ⁇ amyloid ⁇ -peptides
  • a ⁇ 42 amyloid- ⁇ -peptide 1-42
  • Histone Acetyltransferases are involved in histone acetylation (leading to gene activation), chromosome decondensation, DNA repair and non-histone substrate modification
  • WO2004/053140 discloses different compounds as modulators of histone acetyltransferases.
  • the compounds of the present application are the following compounds 6-9:
  • An aspect of the invention provides a method for screening compounds selected from the compounds 6-9 to treat conditions associated with accumulated amyloid-beta peptide deposits.
  • the method comprises (a) administering a HAT Activator compound selected from the compounds 6-9 to an animal model of amyloid-beta peptide deposit accumulation; and (b) selecting a HAT Activator compound selected from the compounds 6-9 that can modulate histone acetylation after administration of the HAT Activator compound in an animal model of amyloid-beta peptide deposit accumulation.
  • An aspect of the invention further provides a method for identifying a histone acetyltransferase (HAT) activator compound selected from the compounds 6-9 to treat conditions associated with accumulated amyloid-beta peptide deposits, wherein the method comprises selecting a HAT Activator compound selected from the compounds 6-9 having one or more of the following features: (a) the EC 50 of the compound is no more than about 1000 nM; (b) the histone acetylation activity in vitro targets histone protein H2, H3, and/or H4; (c) the compound penetrates the blood brain barrier; (d) or a combination thereof.
  • the compound has a molecular mass less than about 500 Da, has a polar surface area less than about 90 ⁇ 2 , has less than 8 hydrogen bonds, or a combination thereof, in order to penetrate the blood brain barrier.
  • An aspect of the invention provides the use of the compounds 6-9 in a method for reducing amyloid beta (A ⁇ ) protein deposits in a subject wherein the method comprises administering to the subject an effective amount of a composition comprising a HAT Activator compound selected from the compounds 6-9 thereby decreasing A ⁇ protein deposits in the subject.
  • the subject exhibits abnormally elevated levels of amyloid beta plaques.
  • the subject is afflicted with Alzheimer's disease, Lewy body dementia, inclusion body myositis, or cerebral amyloid angiopathy.
  • the A ⁇ protein deposit comprises an A ⁇ 40 isomer, an A ⁇ 42 isomer, or a combination of isomers. .
  • the effective amount is at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, at least about 10 mg/kg body weight, at least about 15 mg/kg body weight, at least about 20 mg/kg body weight, at least about 25 mg/kg body weight, at least about 30 mg/kg body weight, at least about 40 mg/kg body weight, at least about 50 mg/kg body weight, at least about 75 mg/kg body weight, or at least about 100 mg/kg body weight.
  • the composition crosses the blood brain barrier.
  • the compound increases histone acetylation.
  • histone acetylation comprises acetylation of histones H2B, H3, H4, or a combination thereof.
  • histone acetylation comprises acetylation of histone lysine residues H3K4, H3K9, H3K14, H4K5, H4K8, H4K12, H4K16, or a combination thereof.
  • An aspect of the invention further provides the use of the compounds 6-9 in a method for treating Alzheimer's Disease in a subject, the method comprising administering to a subject a therapeutic amount of a pharmaceutical composition comprising a HAT activator compound.
  • the effective amount is at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, at least about 10 mg/kg body weight, at least about 15 mg/kg body weight, at least about 20 mg/kg body weight, at least about 25 mg/kg body weight, at least about 30 mg/kg body weight, at least about 40 mg/kg body weight, at least about 50 mg/kg body weight, at least about 75 mg/kg body weight, or at least about 100 mg/kg body weight.
  • the composition crosses the blood brain barrier.
  • the compound increases histone acetylation.
  • histone acetylation comprises acetylation of histones H2B, H3, H4, or a combination thereof.
  • histone acetylation comprises acetylation of histone lysine residues H3K4, H3K9, H3K14, H4K5, H4K8, H4K12, H4K16, or a combination thereof.
  • An aspect of the invention further provides the use of the compounds 6-9 in a method for treating Alzheimer's Disease in a subject, the method comprising administering to a subject a therapeutic amount of a pharmaceutical composition comprising a compound selected from the compounds 6-9.
  • the effective amount is at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, at least about 10 mg/kg body weight, at least about 15 mg/kg body weight, at least about 20 mg/kg body weight, at least about 25 mg/kg body weight, at least about 30 mg/kg body weight, at least about 40 mg/kg body weight, at least about 50 mg/kg body weight, at least about 75 mg/kg body weight, or at least about 100 mg/kg body weight.
  • the composition crosses the blood brain barrier.
  • histone acetylation comprises acetylation of histones H2B, H3, H4, or a combination thereof.
  • histone acetylation comprises acetylation of histone lysine residues H3K4, H3K9, H3K14, H4K5, H4K8, H4K12, H4K16, or a combination thereof.
  • Another aspect of the invention provides the use of the compounds 6-9 in a method for increasing memory retention in a subject afflicted with a neurodegenerative disease, the method comprising administering to a subject a therapeutic amount of a pharmaceutical composition comprising a compound selected from the compounds 6-9. .
  • the neurodegenerative disease comprises Adrenoleukodystrophy (ALD), Alcoholism, Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis (Lou Gehrig's Disease), Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjögren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, Familial fatal insomnia, Frontotemporal lobar degeneration, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy body dementia, Neuroborreliosis, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple System Atrophy, Multiple sclerosis, Narcolepsy, Niemann Pick disease, Parkinson's disease, Pelizaeus-Merzbacher Disease
  • the compound is YF2.
  • the effective amount is at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, at least about 10 mg/kg body weight, at least about 15 mg/kg body weight, at least about 20 mg/kg body weight, at least about 25 mg/kg body weight, at least about 30 mg/kg body weight, at least about 40 mg/kg body weight, at least about 50 mg/kg body weight, at least about 75 mg/kg body weight, or at least about 100 mg/kg body weight.
  • the composition crosses the blood brain barrier.
  • the compound increases histone acetylation.
  • histone acetylation comprises acetylation of histones H2B, H3, H4, or a combination thereof.
  • histone acetylation comprises acetylation of histone lysine residues H3K4, H3K9, H3K14, H4K5, H4K8, H4K12, H4K16, or a combination thereof.
  • An aspect of the invention further provides the use of the compounds 6-9 in a method for increasing synaptic plasticity in a subject afflicted with a neurodegenerative disease, the method comprising administering to a subject a therapeutic amount of a composition that increases histone acetylation in the subject, wherein the composition comprises a compound selected from the compounds 6-9.
  • the effective amount is at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, at least about 10 mg/kg body weight, at least about 15 mg/kg body weight, at least about 20 mg/kg body weight, at least about 25 mg/kg body weight, at least about 30 mg/kg body weight, at least about 40 mg/kg body weight, at least about 50 mg/kg body weight, at least about 75 mg/kg body weight, or at least about 100 mg/kg body weight.
  • the composition crosses the blood brain barrier.
  • the neurodegenerative disease comprises Adrenoleukodystrophy (ALD), Alcoholism, Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis (Lou Gehrig's Disease), Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjögren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, Familial fatal insomnia, Frontotemporal lobar degeneration, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy body dementia, Neuroborreliosis, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple System Atrophy, Multiple sclerosis, Narcolepsy, Niemann Pick disease, Parkinson's
  • ALD Adren
  • synaptic plasticity comprises learning, memory, or a combination thereof.
  • synaptic plasticity comprises long term potentiation (LTP).
  • the compound increases histone acetylation.
  • histone acetylation comprises acetylation of histones H2B, H3, H4, or a combination thereof.
  • histone acetylation comprises acetylation of histone lysine residues H3K4, H3K9, H3K14, H4K5, H4K8, H4K12, H4K16, or a combination thereof.
  • An aspect of the invention further provides the use of the compounds 6-9 in a method for treating Alzheimer's Disease in a subject, the method comprising administering to a subject a therapeutic amount of a pharmaceutical composition comprising a compound selected from the compounds 6-9.
  • the effective amount is at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, at least about 10 mg/kg body weight, at least about 15 mg/kg body weight, at least about 20 mg/kg body weight, at least about 25 mg/kg body weight, at least about 30 mg/kg body weight, at least about 40 mg/kg body weight, at least about 50 mg/kg body weight, at least about 75 mg/kg body weight, or at least about 100 mg/kg body weight.
  • the composition crosses the blood brain barrier.
  • the compound increases histone acetylation.
  • histone acetylation comprises acetylation of histones H2B, H3, H4, or a combination thereof.
  • histone acetylation comprises acetylation of histone lysine residues H3K4, H3K9, H3K14, H4K5, H4K8, H4K12, H4K16, or a combination thereof.
  • An aspect of the invention provides the use of the compounds 6-9 in a method for ameliorating symptoms of Parkinson's Disease in a subject, the method comprising administering to a subject a therapeutic amount of a pharmaceutical composition comprising a HAT activator compound.
  • the HAT activator compound can be a compound selected from the compounds 6-9.
  • the effective amount is at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, at least about 10 mg/kg body weight, at least about 15 mg/kg body weight, at least about 20 mg/kg body weight, at least about 25 mg/kg body weight, at least about 30 mg/kg body weight, at least about 40 mg/kg body weight, at least about 50 mg/kg body weight, at least about 75 mg/kg body weight, or at least about 100 mg/kg body weight.
  • the composition crosses the blood brain barrier. .
  • the compound increases histone acetylation.
  • histone acetylation comprises acetylation of histones H2B, H3, H4, or a combination thereof.
  • histone acetylation comprises acetylation of histone lysine residues H3K4, H3K9, H3K14, H4K5, H4K8, H4K12, H4K16, or a combination thereof.
  • the symptoms of Parkinson's Disease comprise tremor, bradykinesia, dyskinesia, rigidity, postural instability, dystonia, akathisia, dementia, impaired gross motor coordination, or a combination of the listed symptoms.
  • the postural instability comprises impaired imbalance, impaired coordination, or a combination thereof.
  • An aspect of the invention also provides the use of the compounds 6-9 in a method for treating cancer in a subject, the method comprising administering to a subject a therapeutic amount of a pharmaceutical composition comprising a compound selected from the compounds 6-9.
  • the effective amount is at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, at least about 10 mg/kg body weight, at least about 15 mg/kg body weight, at least about 20 mg/kg body weight, at least about 25 mg/kg body weight, at least about 30 mg/kg body weight, at least about 40 mg/kg body weight, at least about 50 mg/kg body weight, at least about 75 mg/kg body weight, or at least about 100 mg/kg body weight.
  • the composition crosses the blood brain barrier.
  • the cancer comprises B cell lymphoma, colon cancer, lung cancer, renal cancer, bladder cancer, T cell lymphoma, myeloma, leukemia, chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, hematopoietic neoplasias, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkins lymphoma, Hodgkins lymphoma, uterine cancer, renal cell carcinoma, hepatoma, adenocarcinoma, breast cancer, pancreatic cancer, liver cancer, prostate cancer, head and neck carcinoma, thyroid carcinoma, soft tissue sarcoma, ovarian cancer, primary or metastatic melanoma, squamous cell carcinoma, basal cell carcinoma, brain cancer, angiosarcoma, hemangiosarcoma,
  • An aspect of the invention provides the use of the compounds 6-9 in a method for treating Huntington's Disease in a subject, the method comprising administering to a subject a therapeutic amount of a pharmaceutical composition comprising a HAT activator compound.
  • the HAT activator compound can be a compound selected from the compounds 6-9.
  • the effective amount is at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, at least about 10 mg/kg body weight, at least about 15 mg/kg body weight, at least about 20 mg/kg body weight, at least about 25 mg/kg body weight, at least about 30 mg/kg body weight, at least about 40 mg/kg body weight, at least about 50 mg/kg body weight, at least about 75 mg/kg body weight, or at least about 100 mg/kg body weight.
  • the composition crosses the blood brain barrier.
  • the compound increases histone acetylation.
  • histone acetylation comprises acetylation of histones H2B, H3, H4, or a combination thereof.
  • histone acetylation comprises acetylation of histone lysine residues H3K4, H3K9, H3K14, H4K5, H4K8, H4K12, H4K16, or a combination thereof.
  • An aspect of the invention provides the use of the compounds 6-9 in for a method of treating a neurodegenerative disease in a subject, the method comprising administering to a subject a therapeutic amount of a pharmaceutical composition comprising a compound selected from the compounds 6-9, thereby treating the neurodegenerative disease in the subject.
  • the effective amount is at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, at least about 10 mg/kg body weight, at least about 15 mg/kg body weight, at least about 20 mg/kg body weight, at least about 25 mg/kg body weight, at least about 30 mg/kg body weight, at least about 40 mg/kg body weight, at least about 50 mg/kg body weight, at least about 75 mg/kg body weight, or at least about 100 mg/kg body weight.
  • the composition crosses the blood brain barrier.
  • the neurodegenerative disease comprises Adrenoleukodystrophy (ALD), Alcoholism, Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis (Lou Gehrig's Disease), Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjögren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, Familial fatal insomnia, Frontotemporal lobar degeneration, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy body dementia, Neuroborreliosis, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple System Atrophy, Multiple sclerosis, Narcolepsy, Niemann Pick disease, Parkinson's
  • ALD Adren
  • synaptic plasticity comprises learning, memory, or a combination thereof.
  • synaptic plasticity comprises long term potentiation (LTP).
  • the compound increases histone acetylation.
  • histone acetylation comprises acetylation of histones H2B, H3, H4, or a combination thereof.
  • histone acetylation comprises acetylation of histone lysine residues H3K4, H3K9, H3K14, H4K5, H4K8, H4K12, H4K16, or a combination thereof.
  • An aspect of the invention provides the use of the compounds 6-9 in a method of decreasing inclusion bodies in a subject afflicted with a neurodegenerative disorder, the method comprising administering to the subject an effective amount of a composition comprising a HAT Activator compound selected from the compounds 6-9, thereby decreasing inclusion bodies in the subject.
  • the inclusion bodies comprise beta-amyloid peptides, native and phosphorylated Tau proteins, native and phosphorylated alpha-synuclein, lipofuscin, cleaved TARDBP (TDB-43), or a combination thereof.
  • the subject exhibits abnormally elevated levels of amyloid beta plaques.
  • the beta-amyloid peptides comprises an A ⁇ 40 isomer, an A ⁇ 42 isomer, or a combination of isomers.
  • the effective amount is at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, at least about 10 mg/kg body weight, at least about 15 mg/kg body weight, at least about 20 mg/kg body weight, at least about 25 mg/kg body weight, at least about 30 mg/kg body weight, at least about 40 mg/kg body weight, at least about 50 mg/kg body weight, at least about 75 mg/kg body weight, or at least about 100 mg/kg body weight.
  • the composition crosses the blood brain barrier.
  • the neurodegenerative disease comprises Adrenoleukodystrophy (ALD), Alcoholism, Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis (Lou Gehrig's Disease), Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjögren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, Familial fatal insomnia, Frontotemporal lobar degeneration, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy body dementia, Neuroborreliosis, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple System Atrophy, Multiple sclerosis, Narcolepsy, Niemann Pick disease, Parkinson's
  • ALD Adren
  • synaptic plasticity comprises learning, memory, or a combination thereof.
  • synaptic plasticity comprises long term potentiation (LTP).
  • the compound increases histone acetylation.
  • histone acetylation comprises acetylation of histones H2B, H3, H4, or a combination thereof.
  • histone acetylation comprises acetylation of histone lysine residues H3K4, H3K9, H3K14, H4K5, H4K8, H4K12, H4K16, or a combination thereof.
  • AD Alzheimer's disease
  • a ⁇ ⁇ -amyloid
  • LTP long-term potentiation
  • Memory is known to be modulated by epigenetics through regulation of gene expression.
  • Epigenetics is defined as the mechanism that changes gene expression by 'marking' DNA or its associated proteins, through processes such as DNA methylation and histone (H) modification, without changing the DNA sequence itself [P9] .
  • Modification of histones by, for example, the addition or removal of acetyl or methyl functional groups causes the chromatin structure to open or close, so that the information contained within the DNA is made more or less accessible to transcription factors.
  • deregulation of one of the epigenetic mechanisms might lead to memory disruption. For instance, reduction of histone acetylation causes the chromatin structure to close, so that the information contained within the DNA might be less accessible to transcription factors and memory formation [P9] .
  • HDACs histone deacetylases
  • HATs share a highly conserved motif containing an acetyl-CoA binding site. HATs can be involved in the pathology of cancer, asthma, neurodegenerative diseases and viral infection. This indicates that specific HAT activators are potential tools for pharmacological research and might find therapeutic applications. So far only one group has reported HAT activators. However, their compounds are neither soluble, nor membrane permeant, which makes them not good drug candidates for the treatment for the diseases described above.
  • This invention is about the synthesis of a new class of HAT activators, which have good potency, excellent solubility, reasonable pharmacokinetic profiles and good membrane and Blood-brain-Barrier (BBB) permeability, hence can be used as a new medicine with minimum amount of side effect for patients with neurodegenerative diseases and cancers.
  • BBB Blood-brain-Barrier
  • the invention provides for compounds with histone acetyltransferase activity, HAT activation potency, high selectivity, reasonable pharmacokinetics and good permeability across the blood-brain-barrier (BBB). These compounds may be used to minimize the side effects for AD patients, the third most costly disease in the U.S., such as improving cognition or memory in AD and Alzheimer's-like pathologies, as well as minimize the side effects for subjects afflicted with other neurodegenerative diseases.
  • the compounds of the invention may also be developed as anti-cancer drugs.
  • the invention provides methods for identifying HAT activators that can acetylate histone proteins thus increasing gene expression in a subject resulting in enhanced memory and cognition.
  • the invention provides for the utilization of HAT agonists as memory enhancers in normal subjects (for example, a subject not afflicted with a neurodegenerative disease). In further embodiments, the invention provides for the utilization of HAT agonists as memory enhancers in aging subjects (for example, a subject who is >55 years old). In further embodiments, the invention provides for the utilization of HAT agonists as memory enhancers for other conditions associated with cognitive decrease/impairment.
  • Non-limiting examples of conditions associated with cognitive decrease/impairment include a variety of syndromes associated with mental retardation and syndromes associated with learning disabilities, Parkinson's disease, Pick's disease, a Lewy body disease, amyotrophic lateral sclerosis, Huntington's disease, Creutzfeld-Jakob disease, Down syndrome, multiple system atrophy, neuronal degeneration with brain iron accumulation type I (Hallervorden-Spatz disease), pure autonomic failure, REM sleep behavior disorder, mild cognitive impairment (MCI), cerebral amyloid angiopathy (CAA), mild cognitive deficits, aging, vascular dementias mixed with Alzheimer's disease, a neurodegenerative disease characterized by abnormal amyloid deposition, and any combination thereof.
  • MCI mild cognitive impairment
  • CAA cerebral amyloid angiopathy
  • HAT activators can first be screened or selected based on their possession of certain characteristics, such as having one or more of: an EC 50 no greater than about 100 nM; a histone acetylation activity in vitro; and the ability to penetrate the BBB.
  • the candidate pool of HAT activators to be tested in animal models of neurodegenerative diseases such as, but not limited to, animals that exhibit elevated levels of inclusion bodies, for example A ⁇ accumulation animal models (e.g., animal models of AD), or, for example, a mouse model for Huntington's disease
  • animal models of neurodegenerative diseases such as, but not limited to, animals that exhibit elevated levels of inclusion bodies, for example A ⁇ accumulation animal models (e.g., animal models of AD), or, for example, a mouse model for Huntington's disease
  • a ⁇ accumulation animal models e.g., animal models of AD
  • a mouse model for Huntington's disease can first be screened or selected based on "medicinal chemistry" strategies.
  • a class of structurally related, but nevertheless formally independent scaffolds can be generated to be deemed as HAT activator candidates.
  • Compounds derived from these scaffolds can first be screened and optimized on computational models. Compounds with highest score will be synthesized and tested for potency.
  • a HAT activator compound does not necessarily preclude the possibility that the compound may also be able to inhibit other HATs.
  • Eukaryotic DNA is highly organized and packaged into the nucleus.
  • the organization and packaging are achieved through the addition of proteins, including core histones H2A, H2B, H3 and H4, which form a complex structure, the chromatin, together with DNA (see FIG. 28 ).
  • the modification of core histones is of fundamental importance to conformational changes of the chromatin.
  • the level of acetylation is related to transcription activity, and then the acetylation induces an open chromatin confirmation that allows the transcription machinery access to promoters.
  • Histone deacetylase HDAC
  • histone acetyltransferase HAT
  • Chromatin acetylation correlates with transcriptional activity (euchromatin)
  • deacetylation correlates with gene silencing.
  • acetylation of H3 in area CA1 of the hippocampus an area in the brain that plays an important role in long-tem memory
  • Histone acetylation and deacetylation are increasingly recognized for their contribution to the tight regulation of gene activation and silencing, respectively. Hence, it is not surprising that deregulation of these mechanisms might lead to the disruption of memoryassociated gene expression, resulting in a number of syndromes associated with mental retardation.
  • Histones The DNA is firstly wrapped around an octamer complex of histones (H) to form nucleosomal units, giving the appearance of beads on a string [B31]. In turn, these nucleosomal units, fold into a higher-order chromatin fiber [B32].
  • H histones
  • Each histoneoctamer complex contains two copies of histones H3 and H4 bordered by two copies of histones 2A and 2B [B32].
  • H1 and its avian variant H5 are linker histones that bind the nucleosome and both the entry and exit sites of the DNA, thus locking the DNA into place and allowing the formation of higher order structure.
  • histone cores and in particular their tails are targets for a considerable number of covalent modifications, such as acetylation, ubiquitination, sumoylation, phosphorylation, citrullination, ADP-ribosylation, and methylation [B33].
  • Histone modifications associated with active gene transcription such as H3 Lys4 methylation and H3 Lys56 acetylation, were found to lead to gene expression.
  • histone modifications associated with the inactivation of gene transcription such as H3 Lys27 methylation and H2A Lys119 ubiquitination were found to cause gene silencing.
  • histone 2B, 3 and 4 because they have been shown to be involved in memory processes [B19, B25].
  • HATs and HDACs Histone modifications and their combinations have been proposed to be involved in gene regulation by modifying the chromatin accessibility and by acting as docking sites for transcription factors and modifying enzymes [B34, B35].
  • One of the most studied histone modifications is the acetylation of the evolutionary-conserved lysine residues on the histone N-termini by histone acetyltransferase (HAT).
  • HAT histone acetyltransferase
  • HDAC histone deacetylation, catalyzed by histone deacetylase
  • the HATs involved in the regulation of gene expression include at least three groups of enzymes [B37] .
  • the general control non-derepressible 5 (Gcn5) is the founding member of the Gcn5 N-acetyltransferases (GNATs).
  • GNATs Gcn5 N-acetyltransferases
  • the GNAT family members include Gcn5, PCAF, Elp3, HAT1m Hpa2 and Nut1.
  • the MYST family is named after the founding members of the family: Morf, Ybf2, Sas2 and Tip60 [B37].
  • other proteins including CBP/p300, Taf1 and a number of nuclear receptor co-activators have been shown to possess intrinsic HAT activity. However, these proteins do not contain a consensus domain and therefore represent an 'orphan class' of HAT enzymes [B37].
  • HDACs form repressor complexes with transcription activators and with other HDACs [B38].
  • Mammalian HDACs can be divided into the classical and the silent information regulator 2 (Sir2)-related protein (sirtruin) families [B39].
  • members of the classical family have another subdivision, which include class I, II and IV, that share sequence similarity and require Zn+ for deacetylase activity.
  • Class I HDACs HDAC 1-3, HDAC8 are related to the yeast gene repressor Rpd3p, and are subunits of at least two distinct co-repressor complexes, the Sin3 complex and the NuRD complex.
  • Class II HDACs are similar to the yeast Hdalp HDAC, they act as gene repressors and have been implicated in various roles in cell differentiation and development.
  • Class IV comprises HDAC11, which has some features of both class I and II HDACs.
  • the sirtruin family includes class III HDACs (SIRT1-7), which are similar to yeast Sir2.
  • Class III HDACs are biochemically and structurally distinct from the classical family and require NAD + as a cofactor. HDACs appear to be involved in gene silencing and heterochromatin formation at centromeres and telomeres (for a review see [B40]).
  • HATs Histone Acetyltransferases
  • HDAC Histone Deacytylases
  • HATs include, but are not limited to GCN5, GCN5L, PCAF, HAT1, ELP3, HPA2, ESA1, SAS2, SAS3, TIP60, HBO1, MOZ, MORF, MOF, SRC1, SRC3, TIF2, GRIP1, ATF-2 [see Lee and Workman (2007) Nat Rev Mol Cell Biol., 8(4):284-95 , Marmorstein (2001) J Molec Biol. 311: 433-444 ; and Kimura et al., (2005) J Biochem. 138(6): 647-662 , which are each hereby incorporated by reference in their entireties].
  • the HAT activator compound of the invention is directed to GCN5, GCN5L, HAT1, PCAF, or a combination thereof.
  • the HAT activator compound of the invention is directed to proteins that possess intrinsic HAT activity, such as nuclear receptor co-activators (for example, CBP/p300 and Tafl).
  • nuclear receptor co-activators for example, CBP/p300 and Tafl.
  • the acetylation of H2, H3, and/or H4 histones is increased.
  • the HAT Activator compound is YF2, depicted in FIG. 3 .
  • HAT activators can be a useful drug candidate with a role similar to HDACi. However, previously available HAT activators had little solubility and membrane permeability, making them unsuitable as drugs.
  • HDACi are in trials for cancer some of which are, for example, 4SC-202 (Nycomed, Germany), which is in a Preclinical stage; AR-42 (Arno therapeutics, Parsippany, NJ) which is in a Preclinical stage; Belinostat (TopoTarget, Rockaway, NJ) which is in Phase II clinical trials; and Entinostat (Bayer Schering) which is in Phase II clinical trials.
  • HDAC inhibitors include Vorinostat, Depsipeptide, and MGCD0103.
  • HDAC inhibitors in clinical use or development are discussed, which include hydroxamic acid compounds (e.g., Vorinostat, Trichostatin A, LAQ824, Panobinostat, Belinostat, and ITF2357), cyclic tetrapeptide compounds (e.g., Depsipeptide), benzamide compounds (e.g., Entinostat and MGCD0103), and short-chain aliphatic acid compounds (e.g., valproic acid, phenyl butyrate, and pivanex).
  • hydroxamic acid compounds e.g., Vorinostat, Trichostatin A, LAQ824, Panobinostat, Belinostat, and ITF235
  • cyclic tetrapeptide compounds e.g., Depsipeptide
  • benzamide compounds e.g., Entinostat and MGCD0103
  • short-chain aliphatic acid compounds e.g., valproic acid, phenyl buty
  • HDACi are or were being developed for neurological diseases, such as an HDACi from Merck (Whitehouse Station, NJ) that is being used for the treatment of neurodegenerative diseases; and HDACi from TopoTarget (Rockaway, NJ) that was being used for the treatment of Huntington's disease, now discontinued; isovaleramide NPS-1776 (NPS Pharmaceutical, Bedminster, New Jersey) that was being used for bipolar disorder, epilepsy, and migraines, now discontinued; and a histone acetyltransferase inhibitor for cancer from TopoTarget A/S (K ⁇ benhavn, Denmark), which was discontinued in the preclinical stage.
  • HAT activator compound of the invention YF2
  • FIG. 3 can be used as adjuvant therapy in several cancers, psychiatric and neurodegenerative diseases and may improve efficacy and safety of treatment for these disorders.
  • the HAT activator compound, YF2 has a moiety which was not mentioned in the abovereferenced patent applications that significantly improves the solubility and membrane and Blood-brain-Barrier (BBB) permeability. See Abel and Zukin (2008) Current Opinion in Pharmacology 8:57-64 ; and Lee and Workman (2007) Nat Rev Mol Cell Biol 8:284-295 .
  • a HAT activator compound can be used to treat a cancer in a subject in need thereof.
  • cancers include B cell lymphoma, colon cancer, lung cancer, renal cancer, bladder cancer, T cell lymphoma, myeloma, leukemia, chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, hematopoietic neoplasias, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkins lymphoma, Hodgkins lymphoma, uterine cancer, renal cell carcinoma, hepatoma, adenocarcinoma, breast cancer, pancreatic cancer, liver cancer, prostate cancer, head and neck carcinoma, thyroid carcinoma, soft tissue sarcoma, ovarian cancer, primary or metastatic melanoma, squamous cell carcinoma, basal cell carcinoma, brain cancer, an
  • a HAT activator compound can be used to treat a neurodegenerative disease in a subject in need thereof.
  • neurodegenerative diseases include Adrenoleukodystrophy (ALD), Alcoholism, Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis (Lou Gehrig's Disease), Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjögren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, Familial fatal insomnia, Frontotemporal lobar degeneration, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, Lewy body dementia, Neuroborreliosis, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple System Atrophy, Multiple
  • FIG. 23 A schematic representation of the processes involved in gene transcription and memory is shown in FIG. 23 .
  • CBP functions as a co-activator that facilitates interactions with the basal transcription machinery and through its HAT activity catalyzes acetylation of the histones, causing a loss in chromosomal repression and increase in the transcription of memory associated genes.
  • mutations in HAT domain CBP were found to cause LTM impairment. For instance, Korzus et a l . demonstrated that inducible dominant-negative CBP mice, with no CBP HAT activity, exhibited normal short-tem memory while LTM was impaired [B8]. Moreover, the impaired LTM was rescued by suppression of the transgene expression and by HDAC inhibitor administration [B8].
  • HDAC inhibition may be beneficial in certain neurodegenerative disorders, such as Huntington's disease, spinal muscular atrophy, amyotrophic lateral sclerosis, ischemia and Rubinstein-Taybi syndrome [B19, B25, B41, B42].
  • HDAC inhibition may provide a therapeutic avenue for memory impairment in neurodegenerative diseases characterized by cognitive disorders such as AD.
  • WT PS1 wild-type PS1 stimulates the transcriptional ability of CBP and p300, whereas the AD associated mutant of PS1 (M146L) does not have this effect [B18, B47].
  • WT PS1 a response to WT PS1 was observed with a construct containing the 721-1679 region of CBP, which contains the CBP acetyltransferase domain [B18].
  • CBP loss and histone deacetylation takes place during neuronal death, which was induced by an APP-directed antibody in primary cortical neurons [B48].
  • Alzheimer's Disease An example of a Neurodegenerative Disease
  • LTM and synaptic plasticity rely on gene expression after an early induction phase characterized by the activation of a number of pathways (for a review, see [B30]). More recently, a fine regulation of memory-related genes and long-term synaptic plasticity has been discovered to involve epigenetic factors [B6]. Indeed, epigenetic modifications, such as DNA methylation and histone post-translational modifications, profoundly affect the ability of polymerases to interact with the open reading frame of DNA without changing the DNA sequence itself. Hence, it would not be surprising that deregulation of epigenetic mechanisms might lead to the disruption of memoryassociated gene expression and synaptic plasticity [B6], contributing to the pathogenesis of diseases characterized by cognitive disorders, such as AD.
  • LTM long-term memory
  • B4 synthesis of new proteins
  • B5 structural changes of the synapse
  • B6 epigenetics
  • N-terminal tails of histone proteins are known to undergo posttranslational modifications, such as histone acetylation, ubiquitination, sumoylation, phosphorylation, citrullination, ADP-ribosylation, and methylation that can dictate the transitions between transcriptionally active or transcriptionally silent chromatin states [B7].
  • posttranslational modifications such as histone acetylation, ubiquitination, sumoylation, phosphorylation, citrullination, ADP-ribosylation, and methylation that can dictate the transitions between transcriptionally active or transcriptionally silent chromatin states [B7].
  • AD Alzheimer's disease
  • a ⁇ Selkoe, D.J. Alzheimer's disease is a synaptic failure. Science (New York, N.Y298, 789-791 (2002 )).
  • a ⁇ -induced reduction in long-term-potentiation (LTP) a physiological correlate of synaptic plasticity that is thought to underlie learning and memory, and phosphorylation of the memory transcription factor CREB, are ameliorated by nitric oxide (NO) donors and cGMP-analogs ( Puzzo, D., et al.
  • Amyloid-beta peptide inhibits activation of the nitric oxide/cGMP/cAMP-responsive element-binding protein pathway during hippocampal synaptic plasticity. JNeurosci 25, 6887-6897 (2005 )). Vice-versa, genetic ablation of NO-synthase 2 (NOS2) results in worsening of the AD phenotype in mice expressing mutated amyloid precursor protein (APP) ( Colton, C.A., et al. NO synthase 2 (NOS2 ) deletion promotes multiple pathologies in a mouse model of Alzheimer's disease. Proceedings of the National Academy of Sciences of the United States of America 103, 12867-12872 (2006 )). These findings show that up-regulation of the NO pathway can be protective in AD.
  • NOS2 mutated amyloid precursor protein
  • AD Alzheimer's disease
  • a ⁇ ⁇ -amyloid
  • LTP long-term potentiation
  • a ⁇ is the proteolytic product of a larger precursor protein, the amyloid precursor protein (APP), which in its mutant form has been found to be implicated in familial AD (FAD) [A9] .
  • PS1 presenilin 1
  • PS2 presenilin 2
  • AD is characterized neuropathologically by neuronal loss, extracellular senile plaques (SPs) and intracellular neurofibrillary tangles (NFTs). SPs are chiefly comprised of A ⁇ aggregates. The major component of NFTs is the microtubule binding protein tau.
  • SPs extracellular senile plaques
  • NFTs neurofibrillary tangles
  • Clinically, AD is characterized by cognitive dysfunction and begins as a synaptic disorder that involves progressively larger areas of the brain over time [S1].
  • An emerging view of the processes involved in synaptic impairment shows that the subtlety and variability of the earliest amnesic symptoms, occurring in the absence of any other clinical signs of brain injury, can be due to discrete changes in the function of a single synapse, produced at least in part, by A ⁇ [S5, S7, S10, S11].
  • synapses An important target for developing a causal therapy for Alzheimer's disease is represented by synapses. Synaptic alterations are highly correlated with the severity of clinical dementia [S1, S2], whereas other important variables such as senile plaques and neurofibrillary tangles are involved to a lesser extent [S1].
  • the importance of synaptic alterations in AD has been confirmed by studies of transgenic (Tg) mouse models of AD [S3] as well as of long-term potentiation (LTP), a widely studied cellular model of learning and memory (L&M) [S4], which is impaired following application of amyloid- ⁇ (A ⁇ ) both in slices and in vivo [S3, S5-S12]. A ⁇ has been found to markedly inhibit LTP. Electrophysiological studies using Tg, human A ⁇ producing mice have often revealed significant deficits in basal synaptic transmission and/or LTP in the hippocampus [S23-S30].
  • Epigenetics is defined as the mechanism that changes gene expression by 'marking' DNA or its associated proteins, through processes such as DNA methylation and histone modification, without changing the DNA sequence itself [A14] . Modification of histones by, for example, the addition or removal of acetyl or methyl functional groups causes the chromatin structure to open or close, so that the information contained within the DNA is made more or less accessible to transcription factors. Hence, it is not surprising that deregulation of one of the epigenetic mechanisms might lead to disruption of memory associated gene expression.
  • Studies of the mechanisms underlying synaptic and memory dysfunction in AD have indicated central roles for the transcription factor CREB (CRE binding protein) and the coactivator CREB binding protein (CBP).
  • AD Alzheimer's disease
  • CRE binding protein transcription factor CREB
  • CBP coactivator CREB binding protein
  • LTP long-term potentiation
  • NO is a central molecule in cellular biochemical processes.
  • the gas has been established as an important messenger molecule in various steps of brain physiology, from development to synaptic plasticity and learning and memory.
  • NO has been found to have a protective effect on A ⁇ -induced damage of the nervous system [S38-S40].
  • a ⁇ has been found to impair NO generation by decreasing NMDA receptor signal transduction [S38], by subtracting NADPH availability to NO-synthase (NOS) [S41], or by inhibiting the phosphorylation of the serine-threonine kinase Akt [S42].
  • NOS NO-synthase
  • Akt serine-threonine kinase
  • the invention provides methods for identifying an agent or compound for the treatment of neurodegenerative diseases (such as AD, Huntington's Disease, Parkinson's Disease, other A ⁇ -accumulation related neurodegenerative disorders or diseases characterized by elevated levels of inclusion bodies) that comprise selecting the agent or compound on the basis of having one or more characteristics that make the compound optimized for treating CNS diseases.
  • neurodegenerative diseases such as AD, Huntington's Disease, Parkinson's Disease, other A ⁇ -accumulation related neurodegenerative disorders or diseases characterized by elevated levels of inclusion bodies
  • the characteristics can comprise: an EC 50 no greater than about 100 nM; histone acetylation activity in vitro; the ability to penetrate the BBB; or a combination thereof.
  • the HAT Activator compound is YF2, depicted in FIG. 3 .
  • the invention provides methods for identifying or designing agents or compounds for the treatment of neurodegenerative diseases, treatment of conditions associated with, but not limited to, elevated inclusion bodies, (e.g., conditions associated with A ⁇ , alpha-synuclein, lipofuscin, cleaved TARDBP-TDP-43, and/or Tau protein accumulation), where computer aided-medicinal chemistry methods are used to identify and/or design agents or compounds tailored to satisfy one or more of the characteristics mentioned above and/or to suit the strengths of various bioassays described herein.
  • elevated inclusion bodies e.g., conditions associated with A ⁇ , alpha-synuclein, lipofuscin, cleaved TARDBP-TDP-43, and/or Tau protein accumulation
  • computer aided-medicinal chemistry methods are used to identify and/or design agents or compounds tailored to satisfy one or more of the characteristics mentioned above and/or to suit the strengths of various bioassays described herein.
  • the invention generally provides methods for identifying compounds which can be used for treating a neurodegenerative disease in a subject.
  • the invention provides methods for identifying compounds which can be used for treating subjects that exhibit abnormally elevated amyloid beta plaques, or elevated Tau protein levels, or elevated alpha-synuclein levels, or inclusions, or lipofuscin level or inclusions, or cleaved TARDBP-TDP-43 level or inclusion, or accumulation of cleaved TARDBP / TDP-43 inclusions.
  • the invention provides methods for identifying compounds which can be used for the treatment of Alzheimer's disease, Lewy body dementia, inclusion body myositis, cerebral amyloid angiopathy, Huntington's Disease, Parkinson's Disease, and cancer.
  • the methods can comprise the identification of test compounds or agents (e.g., peptides (such as antibodies or fragments thereof), small molecules, nucleic acids (such as siRNA or antisense RNA), or other agents) that can bind to a HAT polypeptide molecule and/or activate or enhance the biological activity of a HAT polypeptide or its expression.
  • the compound is a HAT activator (for example a HAT activator compound having Formula (I), (II), (III), (IV), (V), or (VI).
  • the HAT Activator compound is YF2, depicted in FIG. 3 .
  • modulate refers to a change in the activity or expression of a protein molecule. For example, modulation can cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of a secretase protein molecule.
  • a HAT activator compound can be a peptide fragment of a HAT protein that binds to a histone acetyltransferase protein.
  • the HAT activator molecule can encompass any portion of at least about 8 consecutive amino acids of SEQ ID NO: 1, 3, or 5.
  • the fragment can comprise at least about 10 amino acids, a least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, a least about 50 amino acids, at least about 60 amino acids, or at least about 75 amino acids of SEQ ID NO: 1, 3, or 5.
  • the peptide fragment is directed to a HAT protein, such as GCN5, GCN5L, HAT1, or PCAF.
  • HAT1 The polypeptide sequence of a HAT protein, human HAT1, is depicted in SEQ ID NO: 1.
  • the nucleotide sequence of human HAT1 is shown in SEQ ID NO: 2.
  • Sequence information related to HAT1 is accessible in public databases by GenBank Accession numbers NM_003642 (for mRNA) and NP 003633 (for protein).
  • HAT1 is also known as KAT1 (K(lysine) acetyltransferase 1).
  • the protein encoded by this gene is a type B histone acetyltransferase (HAT) that is involved in the rapid acetylation of newly synthesized cytoplasmic histones, which are in turn imported into the nucleus for de novo deposition onto nascent DNA chains.
  • Histone acetylation, particularly of histone H4 plays an important role in replication-dependent chromatin assembly.
  • SEQ ID NO: 1 is the human wild type amino acid sequence corresponding to the HAT protein, the HAT1 enzyme (residues 1-419):
  • SEQ ID NO: 2 is the human wild type nucleotide sequence corresponding to HAT protein, the HAT1 enzyme (residues 1-1682), wherein the underscored ATG denotes the beginning of the open reading frame:
  • the polypeptide sequence of a HAT protein, human PCAF is depicted in SEQ ID NO: 3.
  • the nucleotide sequence of human PCAF is shown in SEQ ID NO: 4.
  • Sequence information related to PCAF is accessible in public databases by GenBank Accession numbers NM_003884 (for mRNA) and NP 003875 (for protein).
  • PCAF is also known as KAT2B (K(lysine) acetyltransferase 2B).
  • CBP and p300 are large nuclear proteins that bind to many sequence-specific factors involved in cell growth and/or differentiation, including c-jun and the adenoviral oncoprotein E1A. The protein encoded by this gene associates with p300/CBP.
  • SEQ ID NO: 3 is the human wild type amino acid sequence corresponding to the HAT protein, the PCAF enzyme (residues 1-832):
  • SEQ ID NO: 4 is the human wild type nucleotide sequence corresponding to HAT protein, the PCAF enzyme (residues 1-4824), wherein the underscored ATG denotes the beginning of the open reading frame:
  • the polypeptide sequence of a HAT protein, human GCN5L is depicted in SEQ ID NO: 5.
  • the nucleotide sequence of human GCN5L is shown in SEQ ID NO: 6.
  • Sequence information related to GCN5L is accessible in public databases by GenBank Accession numbers NM_021078 (for mRNA) and NP_066564.2 (for protein).
  • GCN5L is also known as KAT2A (K(lysine) acetyltransferase 2A).
  • KAT2A, or GCN5 is a histone acetyltransferase (HAT) that functions primarily as a transcriptional activator.
  • SEQ ID NO: 5 is the human wild type amino acid sequence corresponding to the HAT protein, the GCN5L enzyme (residues 1-837):
  • SEQ ID NO: 6 is the human wild type nucleotide sequence corresponding to HAT protein, the GCN5L enzyme (residues 1-3127), wherein the underscored ATG denotes the beginning of the open reading frame:
  • Fragments include all possible amino acid lengths between and including about 8 and 100 about amino acids, for example, lengths between about 10 and 100 amino acids, between about 15 and 100 amino acids, between about 20 and 100 amino acids, between about 35 and 100 amino acids, between about 40 and 100 amino acids, between about 50 and 100 amino acids, between about 70 and 100 amino acids, between about 75 and 100 amino acids, or between about 80 and 100 amino acids.
  • These peptide fragments can be obtained commercially or synthesized via liquid phase or solid phase synthesis methods ( Atherton et al., (1989) Solid Phase Peptide Synthesis: a Practical Approach. IRL Press, Oxford, Engl and).
  • the HAT peptide fragments can be isolated from a natural source, genetically engineered, or chemically prepared. These methods are well known in the art.
  • a HAT Activator compound can also be a protein, such as an antibody (monoclonal, polyclonal, humanized, and the like), or a binding fragment thereof, directed against a histone acetyltransferase enzyme, such as GCN5, GCN5L, PCAF, or HAT1.
  • An antibody fragment can be a form of an antibody other than the full-length form and includes portions or components that exist within full-length antibodies, in addition to antibody fragments that have been engineered.
  • Antibody fragments can include, but are not limited to, single chain Fv (scFv), diabodies, Fv, and (Fab') 2 , triabodies, Fc, Fab, CDR1, CDR2, CDR3, combinations of CDR's, variable regions, tetrabodies, bifunctional hybrid antibodies, framework regions, constant regions, and the like (see, Maynard et al., (2000) Ann. Rev. Biomed. Eng. 2:339-76 ; Hudson (1998) Curr. Opin. Biotechnol. 9:395-402 ).
  • Antibodies can be obtained commercially, custom generated, or synthesized against an antigen of interest according to methods established in the art ( Janeway et al., (2001) Immunobiology, 5th ed., Garland Publishing ).
  • RNA encoding a HAT protein can effectively modulate the expression of a HAT gene (e.g., GCN5, GCN5L, PCAF, or HAT1) from which the RNA is transcribed.
  • Inhibitors are selected from the group comprising: siRNA, interfering RNA or RNAi; dsRNA; RNA Polymerase III transcribed DNAs; ribozymes; and antisense nucleic acid, which can be RNA, DNA, or artificial nucleic acid.
  • Antisense oligonucleotides act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation.
  • antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the DNA sequence encoding a HAT polypeptide can be synthesized, e.g., by conventional phosphodiester techniques ( Dallas et al., (2006) Med. Sci. Monit. 12(4):RA67-74 ; Kalota et al., (2006) Handb. Exp. Pharmacol. 173:173-96 ; Lutzelburger et al., (2006) Handb. Exp. Pharmacol. 173:243-59 ).
  • siRNA comprises a double stranded structure containing from about 15 to about 50 base pairs, for example from about 21 to about 25 base pairs, and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA within the cell.
  • Antisense nucleotide sequences include, but are not limited to: morpholinos, 2'-O-methyl polynucleotides, DNA, RNA and the like.
  • RNA polymerase III transcribed DNAs contain promoters, such as the U6 promoter. These DNAs can be transcribed to produce small hairpin RNAs in the cell that can function as siRNA or linear RNAs that can function as antisense RNA.
  • the HAT activator compound can contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that the target RNA and/or gene is inhibited.
  • these forms of nucleic acid can be single, double, triple, or quadruple stranded, (see for example Bass (2001) Nature, 411, 428 429 ; Elbashir et al., (2001) Nature, 411, 494 498 ; and PCT Publication Nos. WO 00/44895 , WO 01/36646 , WO 99/32619 , WO 00/01846 , WO 01/29058 , WO 99/07409 , WO 00/44914 ).
  • a HAT Activator compound can be a small molecule that binds to a histone acetyltransferase enzyme, such as GCN5, GCN5L, PCAF, or HAT1, and disrupts its function.
  • Small molecules are a diverse group of synthetic and natural substances generally having low molecular weights. They can be isolated from natural sources (for example, plants, fungi, microbes and the like), are obtained commercially and/or available as libraries or collections, or synthesized.
  • Candidate small molecules that interact with a HAT protein can be identified via in silico screening or high-through-put (HTP) screening of combinatorial libraries.
  • Identification and screening antagonists can be further facilitated by determining structural features of the protein, e.g., using X-ray crystallography, neutron diffraction, nuclear magnetic resonance spectrometry, and other techniques for structure determination. These techniques provide for the rational design or identification of antagonists, in addition to protein agonists.
  • the invention provides methods for screening and identifying compounds useful for treating a neurodegenerative disease in a subject.
  • the invention provides methods for identifying compounds which can be used for treating subjects that exhibit, for example, abnormally elevated amyloid beta plaques, or elevated Tau protein levels, or elevated alpha-synuclein levels, or inclusions, or lipofuscin level or inclusions, or cleaved TARDBP-TDP-43 level or inclusion, or accumulation of cleaved TARDBP / TDP-43 inclusions, or a combination thereof.
  • the method comprises selecting a HAT Activator compound that comprises one or both of the following features: (a) the EC 50 of the compound is no more than about 1000 nM; (b) the compound penetrates the blood brain barrier; (c) the compound enhances histone acetylation (for example acetylates histone protein H3 or H4), or a combination thereof.
  • the compound for example the HAT Activator, has an EC 50 of at least about 0.1 nM, at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 25 nM, at least about 50 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, or at least about 900 nM.
  • the HAT Activator compound can have a molecular mass less than about 500 Da in order to penetrate the blood brain barrier.
  • the HAT Activator compound can have a polar surface area less than about 90 ⁇ 2 and should have 8 or fewer hydrogen bonds in order to penetrate the blood brain barrier.
  • the screening and identifying of the compound can comprise in silico screening, molecular docking, in vivo screening, in vitro screening, or a combination thereof.
  • Test compounds such as HAT Activator compounds
  • HAT Activator compounds can be screened from large libraries of synthetic or natural compounds (see Wang et al., (2007) Curr Med Chem, 14(2):133-55 ; Mannhold (2006) Curr Top Med Chem, 6 (10):1031-47 ; and Hensen (2006) Curr Med Chem 13(4):361-76 ).
  • Numerous means are currently used for random and directed synthesis of saccharide, peptide, and nucleic acid based compounds.
  • Synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.).
  • a rare chemical library is available from Aldrich (Milwaukee, Wis.).
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g. Pan Laboratories (Bothell, Wash.) or MycoSearch (N.C.), or are readily producible.
  • natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means ( Blondelle et al., (1996) Tib Tech 14:60 ).
  • Libraries of interest in the invention include peptide libraries, randomized oligonucleotide libraries, synthetic organic combinatorial libraries, and the like.
  • Degenerate peptide libraries can be readily prepared in solution, in immobilized form as bacterial flagella peptide display libraries or as phage display libraries.
  • Peptide ligands can be selected from combinatorial libraries of peptides containing at least one amino acid.
  • Libraries can be synthesized of peptoids and non-peptide synthetic moieties. Such libraries can further be synthesized which contain non-peptide synthetic moieties, which are less subject to enzymatic degradation compared to their naturally-occurring counterparts.
  • Libraries are also meant to include for example but are not limited to peptide-on-plasmid libraries, polysome libraries, aptamer libraries, synthetic peptide libraries, synthetic small molecule libraries, neurotransmitter libraries, and chemical libraries.
  • the libraries can also comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more of the functional groups described herein.
  • a combinatorial library of small organic compounds is a collection of closely related analogs that differ from each other in one or more points of diversity and are synthesized by organic techniques using multi-step processes.
  • Combinatorial libraries include a vast number of small organic compounds.
  • One type of combinatorial library is prepared by means of parallel synthesis methods to produce a compound array.
  • a compound array can be a collection of compounds identifiable by their spatial addresses in Cartesian coordinates and arranged such that each compound has a common molecular core and one or more variable structural diversity elements. The compounds in such a compound array are produced in parallel in separate reaction vessels, with each compound identified and tracked by its spatial address. Examples of parallel synthesis mixtures and parallel synthesis methods are provided in U.S. Ser. No.
  • phage display libraries are described in Scott et al., (1990) Science 249:386-390 ; Devlin et al., (1990) Science, 249:404-406 ; Christian, et al., (1992) J. Mol. Biol. 227:711-718 ; Lenstra, (1992) J. Immunol. Meth. 152:149-157 ; Kay et al., (1993) Gene 128:59-65 ; and PCT Publication No. WO 94/18318 .
  • In vitro translation-based libraries include but are not limited to those described in PCT Publication No. WO 91/05058 ; and Mattheakis et al., (1994) Proc. Natl. Acad. Sci. USA 91:9022-9026 .
  • non-peptide libraries such as a benzodiazepine library (see e.g., Bunin et al., (1994) Proc. Natl. Acad. Sci. USA 91:4708-4712 ), can be screened.
  • Peptoid libraries such as that described by Simon et al., (1992) Proc. Natl. Acad. Sci. USA 89:9367-9371 , can also be used.
  • Another example of a library that can be used, in which the amide functionalities in peptides have been permethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (1994), Proc. Natl. Acad. Sci. USA 91:11138-11142 .
  • the three dimensional geometric structure of an active site for example that of a HAT polypeptide can be determined by known methods in the art, such as X-ray crystallography, which can determine a complete molecular structure. Solid or liquid phase NMR can be used to determine certain intramolecular distances. Any other experimental method of structure determination can be used to obtain partial or complete geometric structures.
  • the geometric structures can be measured with a complexed ligand, natural or artificial, which can increase the accuracy of the active site structure determined.
  • a compound that binds to a HAT protein such as GCN5, GCN5L, PCAF, or HAT1
  • GCN5L complexed ligand
  • 1YGH or 2RC4 for at least 20 amino acid residues for the acetyltransferase active site of the HAT protein (the HAT domain), wherein the coordinates have a root mean square deviation therefrom, with respect to at least 50% of C ⁇ atoms, of not greater than about 2 ⁇ , in a computer readable format; (3) converting the atomic coordinates into electrical signals readable by a computer processor to generate a three dimensional model of the HAT protein; (4) performing a data processing method, wherein electronic test compounds from the library are docked onto the three dimensional model of the HAT protein; and determining which test compound fits into the active site of the three dimensional model of the HAT protein, thereby identifying which compound would bind to a HAT protein.
  • the method can further comprise: synthesizing or obtaining the compound determined to dock to the active site of the HAT protein; contacting the HAT protein with the compound under a condition suitable for binding; and determining whether the compound modulates HAT protein expression or mRNA expression, or HAT protein activity using a diagnostic assay.
  • Molecular imprinting for instance, can be used for the de novo construction of macromolecular structures such as peptides that bind to a molecule. See, for example, Kenneth J. Shea, Molecular Imprinting of Synthetic Network Polymers: The De Novo synthesis of Macromolecular Binding and Catalytic Sites, TRIP Vol. 2, No. 5, May 1994 ; Mosbach, (1994) Trends in Biochem. Sci., 19(9 ); and Wulff, G., in Polymeric Reagents and Catalysts (Ford, W. T., Ed.) ACS Symposium Series No.
  • One method for preparing mimics of a HAT protein involves the steps of: (i) polymerization of functional monomers around a known substrate (the template) that exhibits a desired activity; (ii) removal of the template molecule; and then (iii) polymerization of a second class of monomers in, the void left by the template, to provide a new molecule which exhibits one or more desired properties which are similar to that of the template.
  • Other binding molecules such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids, and other biologically active materials can also be prepared.
  • This method is useful for designing various biological mimics that are more stable than their natural counterparts, because they are prepared by the free radical polymerization of functional monomers, resulting in a compound with a nonbiodegradable backbone.
  • Other methods for designing such molecules include , e.g., drug design based on structure activity relationships, which require the synthesis and evaluation of a number of compounds and molecular modeling.
  • the invention also provides in vivo and in vitro methods for identifying a compound that binds to a HAT protein.
  • the method comprises: (a) obtaining a tissue and/or cells that express a HAT protein (such as GCN5, GCN5L, PCAF, or HAT1); (b) contacting the tissue and/or cell with a ligand source for an effective period of time; (c) measuring a secondary messenger response, wherein the response is indicative of a ligand binding to a HAT protein; (d) isolating the ligand from the ligand source; and (e) identifying the structure of the ligand that binds a HAT protein, thereby identifying which compound would bind to a HAT protein.
  • a HAT protein such as GCN5, GCN5L, PCAF, or HAT1
  • ligand source can be any compound library described herein, or a library of neurotransmitters that can be used to screen for compounds that would act as an agonist of a HAT protein (such as GCN5, GCN5L, PCAF, or HAT1). Screening compound libraries listed herein [also see U.S. Patent Application Publication No. 2005/0009163 ,), , in combination with in vivo animal studies and functional assays can be used to identify HAT Activator compounds that can be used to treat subjects afflicted with abnormal A ⁇ deposits, such as AD or to treat cancer.
  • a HAT Activator compound can be a compound that increases the activity and/or expression of a HAT molecule (e.g., GCN5, GCN5L, PCAF, or HAT1) in vivo and/or in vitro.
  • HAT Activator compounds can be compounds that exert their effect on the activity of a HAT protein via the expression, via post-translational modifications, or by other means.
  • a HAT Activator compound can increase HAT protein or mRNA expression, or acetyltransferase activity by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 99%, or 100%.
  • Test compounds or agents which bind to a HAT molecule can be identified by various assays.
  • the assay can be a binding assay comprising direct or indirect measurement of the binding of a test compound or a known HAT ligand to the active site of a HAT protein.
  • the assay can also be an activity assay comprising direct or indirect measurement of the activity of a HAT molecule.
  • the assay can also be an expression assay comprising direct or indirect measurement of the expression of a HAT mRNA or protein.
  • the various screening assays can be combined with an in vivo assay comprising measuring the effect of the test compound on cognitive and synaptic function in an animal model for neurodegenerative disorders, such as, but not imited to, AD or Huntington's Disease.
  • the diagnostic assay of the screening methods of the invention can also involve monitoring the expression of a HAT molecule.
  • inhibitors of the expression of a HAT molecule can be identified via contacting a HAT-positive cell or tissue with a test compound and determining the expression of a HAT protein or HAT mRNA in the cell.
  • the protein or mRNA expression level of a HAT molecule in the presence of the test compound is compared to the protein or mRNA expression level of a HAT protein in the absence of the test compound.
  • the test compound can then be identified as an inhibitor of expression of a HAT protein (such as GCN5, GCN5L, PCAF, or HAT1) based on this comparison.
  • Acivators of the expression of a HAT molecule can also be identified via contacting a HAT-positive cell or tissue with a test compound and determining the expression of a HAT protein or HAT mRNA in the cell.
  • the protein or mRNA expression level of a HAT molecule in the presence of the test compound is compared to the protein or mRNA expression level of a HAT protein in the absence of the test compound.
  • the test compound can then be identified as an activator of expression of a HAT protein (such as GCN5, GCN5L, PCAF, or HAT1) based on this comparison.
  • the compound when expression of HAT protein or mRNA is statistically or significantly more in the presence of the test compound than in its absence, the compound is identified as an activator of the expression of a HAT protein or mRNA.
  • the test compound can also be said to be a HAT Activator compound (such as an agonist).
  • the expression level of a HAT protein or mRNA in cells can be determined by methods described herein.
  • BIA Bimolecular Interaction Analysis
  • the invention provides for compounds that bind to a HAT activator protein, such as GCN5, GCN5L, PCAF, or HAT1. These compounds can be identified by the screening methods and assays described herein, and enhance the activity or expression of HAT activator proteins.
  • the HAT Activator reference compound, YF2 can be synthesized according to the scheme depicted in FIG. 29 .
  • Other analogs of HAT Activator compounds having Formula I can be similarly synthesized. However, only compounds with the formula 6-9 are according to this invention. The rest of the compounds mentioned in those figures are reference compounds.
  • the HAT Activator compound, 10 can be synthesized according to the scheme depicted in FIG. 37 .
  • the HAT Activator compound, 11, can be synthesized according to the scheme depicted in FIG. 39 .
  • the HAT Activator compound, 12 can be synthesized according to the scheme depicted in FIG. 40 .
  • the HAT Activator compound, 13, can be synthesized according to the scheme depicted in FIG. 42 .
  • the HAT Activator compound, 14 can be synthesized according to the scheme depicted in FIG. 41 .
  • the HAT Activator compound, 15, can be synthesized according to the scheme depicted in FIG. 43 .
  • the HAT Activator compound, 16 can be synthesized according to the scheme depicted in FIG. 44 .
  • the HAT Activator compound, 17, can be synthesized according to the scheme depicted in FIG. 45 .
  • the HAT Activator compound, 18, can be synthesized according to the scheme depicted in FIG. 46 .
  • the HAT Activator compound, 19, can be synthesized according to the scheme depicted in FIG. 51 .
  • the HAT Activator compound, 20 can be synthesized according to the scheme depicted in FIG. 38 .
  • the compounds of the invention can be generally synthesized according to the scheme based on the diagram depicted in FIG. 29 .
  • a pharmaceutically acceptable salt of a compound of Formula (I) is an acid addition salt, for example a hydrochloride, sulfate, or phosphate salt.
  • a pharmaceutically acceptable salt of a compound of Formula (I) is a base addition salt, for example a sodium, potassium, calcium, or ammonium salt.
  • the base addition salt is a tetrafluoroboro salt.
  • a pharmaceutically acceptable salt of a compound of Formula (II) is an acid addition salt, for example a hydrochloride, sulfate, or phosphate salt.
  • a pharmaceutically acceptable salt of a compound of Formula (II) is a base addition salt, for example a sodium, potassium, calcium, or ammonium salt.
  • the base addition salt is a tetrafluoroboro salt.
  • a pharmaceutically acceptable salt of a compound of Formula (III) is an acid addition salt, for example a hydrochloride, sulfate, or phosphate salt.
  • a pharmaceutically acceptable salt of a compound of Formula (III) is a base addition salt, for example a sodium, potassium, calcium, or ammonium salt.
  • the base addition salt is a tetrafluoroboro salt.
  • a pharmaceutically acceptable salt of a compound of Formula (IV) is an acid addition salt, for example a hydrochloride, sulfate, or phosphate salt.
  • a pharmaceutically acceptable salt of a compound of Formula (IV) is a base addition salt, for example a sodium, potassium, calcium, or ammonium salt.
  • the base addition salt is a tetrafluoroboro salt.
  • a pharmaceutically acceptable salt of a compound of Formula (V) is an acid addition salt, for example a hydrochloride, sulfate, or phosphate salt.
  • a pharmaceutically acceptable salt of a compound of Formula (V) is a base addition salt, for example a sodium, potassium, calcium, or ammonium salt.
  • the base addition salt is a tetrafluoroboro salt.
  • the invention provides the use of the compounds 6-9 in methods for reducing inclusion bodies (e.g., amyloid beta (A ⁇ ) protein deposits, native and phosphorylated Tau proteins, native and phosphorylated alpha-synuclein, lipofuscin, cleaved TARDBP (TDB-43), or a combination thereof) in a subject afflicted with a neurodegenerative disease (e.g., a AD, Huntington's Disease, or Parkinson's Disease) by administering any one of the HAT Activator compounds 6-9.
  • a neurodegenerative disease e.g., a AD, Huntington's Disease, or Parkinson's Disease
  • the invention also provides the use of the compounds 6-9 in methods for treating a neurodegenerative disease in a subject by administering any one of the HAT Activator compounds 6-9.
  • the invention further provides the use of the compounds 6-9 in methods for treating cancer in a subject by administering any one of the HAT Activator compounds 6-9.
  • the HAT Activator compounds
  • HAT activator compounds selected from the compounds 6-9 are first screened for their ability to satisfy one or more of the following characteristics: an EC 50 no greater than about 100 nM; a histone acetylation activity in vitro; the ability to penetrate the BBB; or a combination thereof.
  • the method comprises administering to the subject an effective amount of a composition comprising a HAT Activator compound.
  • the subject exhibits abnormally elevated amyloid beta plaques, or elevated Tau protein levels, or accumulations of alpha-synuclein, or accumulations of lipofuscin, or accumulation of cleaved TARDBP (TDB-43) levels, or a combination thereof.
  • the A ⁇ protein deposit comprises an A ⁇ 40 isomer, an A ⁇ 42 isomer, or a combination thereof.
  • the subject is afflicted with Alzheimer's disease, Lewy body dementia, inclusion body myositis, Huntington's Disease, Parkinson's Disease, or cerebral amyloid angiopathy.
  • the subject is afflicted with cancer.
  • the dosage administered can be a therapeutically effective amount of the composition sufficient to result in amelioration of symptoms of a neurogenerative disease such as, but not limited to reducing inclusion bodies (e.g., amyloid beta (A ⁇ ) protein deposits, native and phosphorylated Tau proteins, native and phosphorylated alpha-synuclein, lipofuscin, cleaved TARDBP (TDB-43), or a combination thereof), or reducing memory loss in a subject.
  • reducing inclusion bodies e.g., amyloid beta (A ⁇ ) protein deposits, native and phosphorylated Tau proteins, native and phosphorylated alpha-synuclein, lipofuscin, cleaved TARDBP (TDB-43), or a combination thereof
  • reducing inclusion bodies e.g., amyloid beta (A ⁇ ) protein deposits, native and phosphorylated Tau proteins, native and phosphorylated alpha-synuclein, lipofuscin, cleaved TARDBP (TD
  • observing at least, about a 25% reduction, at least about a 30% reduction, at least about a 40% reduction, at least about a 50% reduction, at least about a 60% reduction, at least about a 70% reduction, at least about a 80% reduction, at least about a 85% reduction, at least about a 90% reduction, at least about a 95% reduction, at least about a 97% reduction, at least about a 98% reduction, or a 100% reduction in inclusion bodies or memory loss in a subject is indicative of amelioration of symptoms of a neurogenerative disease (for example, including, but not limited to, AD, Huntington's Disease, Parkinson's Disease).
  • This efficacy in reducing inclusion occurrence can be, for example, a meaure of ameliorating symptoms of a neurogenerative disease.
  • the therapeutically effective amount is at least about 0.1 mg/kg body weight, at least about 0.25 mg/kg body weight, at least about 0.5 mg/kg body weight, at least about 0.75 mg/kg body weight, at least about 1 mg/kg body weight, at least about 2 mg/kg body weight, at least about 3 mg/kg body weight, at least about 4 mg/kg body weight, at least about 5 mg/kg body weight, at least about 6 mg/kg body weight, at least about 7 mg/kg body weight, at least about 8 mg/kg body weight, at least about 9 mg/kg body weight, at least about 10 mg/kg body weight, at least about 15 mg/kg body weight, at least about 20 mg/kg body weight, at least about 25 mg/kg body weight, at least about 30 mg/kg body weight, at least about 40 mg/kg body weight, at least about 50 mg/kg body weight, at least about 75 mg/kg body weight, at least about 100 mg/kg body weight, at least about 200 mg/kg body weight, at least about 250 mg/kg body weight, at
  • a HAT activator compound can be administered to the subject one time (e.g., as a single injection or deposition).
  • a HAT activator compound of the invention can be administered once or twice daily to a subject in need thereof for a period of from about 2 to about 28 days, or from about 7 to about 10 days, or from about 7 to about 15 days. It can also be administered once or twice daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof.
  • the dosage administered can vary depending upon known factors such as the pharmacodynamic characteristics of the active ingredient and its mode and route of administration; time of administration of active ingredient; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired; and rate of excretion.
  • Toxicity and therapeutic efficacy of therapeutic compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Therapeutic agents that exhibit large therapeutic indices are useful.
  • Therapeutic compositions that exhibit some toxic side effects can be used.
  • a therapeutically effective dose of a HAT activator compound can depend upon a number of factors known to those of ordinary skill in the art.
  • the dose(s) of a HAT activator compound for example a compound selected from the compounds 6-9, can vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the HAT activator compound to have upon a HAT protein or a protein exhibiting intrinsic HAT activity. These amounts can be readily determined by a skilled artisan.
  • HAT activator compounds of the invention can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions can comprise a HAT activator compound (e.g., a compound of HAT activator compounds selected from the compounds 6-9, and a pharmaceutically acceptable carrier.
  • the compositions can be administered alone or in combination with at least one other agent, such as a stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.
  • a pharmaceutically acceptable carrier can comprise any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Any conventional media or agent that is compatible with the active compound can be used. Supplementary active compounds can also be incorporated into the compositions.
  • any of the therapeutic applications described herein can be applied to any subject in need of such therapy, including, for example, a mammal such as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human.
  • a mammal such as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EM TM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the HAT Activator compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein.
  • examples of useful preparation methods are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.
  • compositions can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such as sucrose or saccharin
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • Example 1 A HAT Activator compound.
  • Reference compound YF2 a Histone Acetyltransferase (HAT) Activator of the invention ( FIG. 3 ) , is a good drug candidate to ameliorate memory in neurodegenerative diseases (i.e. Alzheimer's disease and Huntington's disease) and treatment for a variety of cancers.
  • neurodegenerative diseases i.e. Alzheimer's disease and Huntington's disease
  • the western blot showed that it not only crosses the BBB, but also increases histone 3 acetylation levels of the hippocampus ( FIG. 1 ).
  • the title compound ameliorates the contextual fear memory deficit in A ⁇ 42 - infused mice ( FIG. 2 ).
  • a ⁇ 42 is a protein that is produced in high amount in AD and is responsible for the impairment of synaptic functions and memory.
  • MOM has medium solubility (DMSO 10% in H 2 O). MOM was administered 25mg/kg to WT mice (i.p.). The mice liver and hippocampus were extracted 1hr after treatment. The liver showed a very slight increase of AcH3, indicating that the drug has either very little efficacy OR very little membrane permeability ( FIG. 6 ). The hippocampus had no increase in AcH3 levels, indicating the drug is either ineffective OR does not cross the blood brain barrier (BBB) ( FIG. 6 ). Although MOM failed to increase AcH3 levels in the hippocampus and liver, the experiment was repeated with a new administration (gavage and i.p. 25mg/kg).
  • BBB blood brain barrier
  • mice fear conditioning treatment of the mice was subsequently carried out to see if the drug is active after induction of learning.
  • the mouse cortex was also extracted. Hippocampus, cortex, and liver samples again showed no increase of AcH3 levels, indicating the drug is either ineffective OR does not cross the BBB OR does not cross the cell membrane ( FIG. 7 ).
  • a reference compound YF2 ( FIG. 3 ), was synthesized. The preparation of YF2 was without a column and 2 phases were visible: clear and oily. YF2 (50 mg/kg, i.p.) was subsequently administered to mice. Two and four hrs after its administration, the mice were sacrificed and hippocampi were extracted. Interestingly, YF2 was able to cross the BBB, penetrate the cells and increase AcH3 (lane 1 vs. lanes 9, 10) ( FIG. 8 ). Given that the compound was not 100% clean and needed to be further purified/verified, we synthesized more YF2 and purified it. Purity was verified through Nuclear Magnetic Resonance (NMR). Mice were administered with YF2 (i.p.
  • Contextual and cued fear conditioning was performed to assess whether the compound is capable of ameliorating amyloid-beta (A ⁇ ) induced memory defect.
  • a ⁇ is a peptide which is elevated in Alzheimer's disease.
  • the hippocampus plays a key role in contextual memory and in Alzheimer's Disease. This type of cognitive test is much faster than other behavioral tasks that require multiple days of training and testing [Q1, Q2].
  • Our conditioning chamber was in a sound-attenuating box. A clear Plexiglas window allowed the experimenter to film the mouse performance with a camera placed on a tripod and connected to the Freezeframe software (MED Ass. Inc.). To provide background white noise (72 dB), a single computer fan was installed in one of the sides of the sound-attenuating chamber.
  • the conditioning chamber had a 36-bar insulated shock grid floor. The floor was removable, and after each experimental subject, we cleaned it with 75% ethanol and then with water. Only one animal at a time was present in the experimentation room.
  • mice were placed in the conditioning chamber for 2 min before the onset of a discrete tone (CS) (a sound that lasted 30 sec at 2800 Hz and 85 dB).
  • CS discrete tone
  • US foot shock
  • mice were given a foot shock (US) of 0.8 mA for 2 sec through the bars of the floor.
  • the mice were left in the conditioning chamber for another 30 sec and were then placed back in their home cages. Freezing behavior, defined as the absence of all movement except for that necessitated by breathing, was scored using the Freezeview software.
  • the shock intensity was increased by 0.1 mV to 0.7 mV and then returned to 0 mV in 0.1 mV increments at 30 sec intervals. Threshold to vocalization, flinching, and then jumping was quantified for each animal by averaging the shock intensity at which each animal manifests a behavioral response to the foot shock.
  • YF2 was i.p. administered to mice (one group of mice was administered with 20 mg/kg, 2hrs before the electric shock, whereas another group was administered with 5 mg/kg, 30 minutes before the electric shock). YF2 at both doses was capable of dramatically increasing the freezing time demonstrating that the compound rescues the defect in contextual memory.
  • the compound alone at the highest concentration (20 mg/kg) did not affect contextual memory ( FIG. 10 ) indicating that the compound per se is not toxic with respect to memory.
  • Cued memory was not changed in the different groups indicating that YF2 does not affect amygdala function ( FIG. 11 ).
  • no difference was observed among different groups of mice in different sets of experiments in which we assessed sensory threshold in the presence of vehicle, YF2 alone, A ⁇ alone, or YF2 plus A ⁇ ( FIG. 12 ).
  • the task is a hybrid of the Morris Water Maze (MWM) and the radial arm land maze. This task is altered in A ⁇ -infused mice.
  • the motivation for the animals is the immersion in water.
  • the mouse needed to swim in 6 alleys (arms) radiating from a central area until it found a hidden (submerged) platform at the end of one of the arms, based on visual cues placed in the room.
  • the goal arm was kept constant for all trials, with a different start arm on successive trials, such that the learning criterion was reached in 2 days.
  • the first day of the protocol was a training day.
  • Mice were trained to identify the platform location by alternating between a visible and a hidden platform in a goal arm.
  • the final 3 trials on day 1 and all 15 trials on day 2 used a hidden escape platform to force mice to use spatial cues to identify the location of the goal arm.
  • the mouse was guided gently through the water by placing a hand behind it to direct it towards the platform. The mouse rested on the platform for 15 sec. After completing the trial, the mouse was removed from the pool, gently towel dried and placed back into its cage under a heat lamp. The goal platform location was different for each mouse.
  • mice from cohort 1 After all the mice in the first cohort have had a trial to locate a visible platform, the platform was switched from visible to hidden. After each mouse from cohort 1 completed six alternating trials between visible and hidden platforms, the mice was left to rest under a heating source, and mice from the second cohort were tested in the same way. After completing the six alternating trials, mice from cohort 2 returned to their cages to rest.
  • mice from the first cohort completed trials 7-12 again using the alternating visible-hidden platform location.
  • mice from the second cohort completed trials 7-12.
  • all mice had performed 3 hidden platform trials.
  • the same procedure was repeated as on day 1 for all 15 trials using only the hidden platform.
  • averages for each mouse were calculated using blocks of 3 trials.
  • vehicle-treated mice exhibit ⁇ 1 error over three trials near the end of the second day.
  • a ⁇ -infused mice failed to learn, making 3-4 errors throughout the training session, with no improvement over trials.
  • Treatment with YF2 [(i.p., 5 mg/kg, 30 min prior to the 1 st trial (for the 1 st group of tests) and 30 min prior to the 7 th trial (for the 2 nd group of tests)] rescued the A ⁇ -induced memory impairment.
  • TSA histone de-acetylation through trichostatin A
  • FC contextual fear conditioning
  • CBP functions as a co-activator that facilitates interactions with the basal transcription machinery by working as an acetyltransferase (HAT) that catalyzes acetylation of the histones, causing a loss in chromosomal repression and increase in the transcription of memory associated genes.
  • HAT acetyltransferase
  • HDACs were found to remove an acetyl group from histones, thus restricting access of the transcriptional machinery to the DNA.
  • HDAC inhibitors have been shown to enhance LTP and contextual fear memory, a form of associative memory in which animals must associate a neutral stimulus with an aversive one[A25].
  • HDAC inhibitor an HDAC III inhibitor
  • RTS Rubinstein-Taybi syndrome
  • Nicotinamide an HDAC III inhibitor
  • SAHA was found to restore cognition in the triple Tg mouse model of AD via a mechanism involving reduction of Thr231-phosphotau [A26].
  • HDAC inhibitors induced sprouting of dendrites, an increased number of synapses, and reinstated learning and access to long-term memories in the CK-p25 Tg mouse model of neuronal loss [A23].
  • HDAC inhibitors could affect neuronal function through a variety of mechanisms including epigenetic and non-epigenetic changes [A27]. Whether cognitive deficits following A ⁇ elevation may be induced by epigenetic modification on histone acetylation (via chromatin remodeling) has not been determined.
  • WT-PS1 stimulates the transcriptional activity ability of CBP whereas its AD M146L mutant did not produce such an effect [A20] indicating that CBP and its HAT activity in AD may be involved.
  • a CBP mutant lacking HAT activity is not capable of responding to WT-PS1 in terms of increased transcription activating ability.
  • CBP and its HAT region appear to be essential for enhancing transcription in vitro following PS1 stimulation.
  • the inventors find that histone acetylation level of APP/PS1 mice is different than in WT mice, thus identifying AD as a disease of epigenetic etiology.
  • AD Alzheimer's disease
  • AD is thought to begin as a synaptic disorder that progressively leads to greater neuronal dysfunction, leading to memory loss [A28].
  • TSA HDAC inhibitor
  • mice 4 month-old mice were divided into 4 groups: APP/PS1 with TSA, APP/PS1 with vehicle, WT with TSA and WT with vehicle. TSA and vehicle control solution were administered i.p. at a concentration of 2 ⁇ g/g body weight. We found a similar shock threshold among the various groups of mice. Then, mice were trained to associate neutral stimuli with an aversive one. They were placed in a novel context (FC box), exposed to a white noise cue paired with a mild foot shock, and injected with TSA 2hrs before training.
  • FC box novel context
  • the APP and PS1 transgenes could affect neuronal function through different mechanisms [A30, A31], including direct effects by A ⁇ .
  • the trafficking and signaling properties of full-length APP and its cleavage products are likely different, which could impact aspects of synaptic function differently.
  • a ⁇ per se is responsible for the deficits observed in our studies on Tg mice. Since it has already been described that natural oligomers of human A ⁇ , in the absence of monomers and fibrils, markedly inhibit LTP in vivo [A6], we will apply 200 nM oligomeric A ⁇ 42 concurrently with TSA (1.65 ⁇ M) for 30 minutes to WT slices prior to inducing LTP.
  • oligomeric A ⁇ 42 should inhibit LTP and fear memory, and demonstrate that TSA reestablishes normal LTP and contextual fear memory following A ⁇ 42 treatment. TSA alone should not have any effect.
  • Controls will be performed using a latent inhibition training paradigm to exclude that changes in CBP HAT activity are due to novel context alone or the electric shock instead of the association between them [A25].
  • animals will be pre-exposed to a novel context prior to receiving the electric shock so that the animal will form a spatial memory that blocks the formation of an associative contextual fear memory.
  • HDAC activity using a new fluorimetric assay on hippocampi using the experimental paradigm as in the HAT assay. Without being bound by theory, these experiments will establish if CBP and/or HDACs are altered following overexpression of the APP and PS1transgenes.
  • HDAC inhibition might rescue the histone 4 acetylation levels defect observed in APP/PS1 mice was also tested. Injection of TSA (2 ⁇ g/g body weight; i.p) 2 hours prior to contextual fear conditioning enhanced H4 acetylation of APP/PS1 mice ( FIG. 22 ). Without being bound by theory, AD is likely to be a disease with an epigenetic motif and HDAC inhibitors can elevate decreased levels of histone 4 in an AD mouse model.
  • mice Double Tg mice will be obtained by crossing APP and PS1 animals (genotyped by PCR) [A29] .
  • APP proliferative protein
  • PS1 proliferative protein
  • Oligomeric A ⁇ 42 will be prepared from commercially available synthetic peptides (American Peptides Co), as described [A33,A34] .
  • CA1 fEPSPs will be recorded by placing stimulating and the recording electrodes in CA1 stratum radiatum. Following BST assessment, a 15min baseline will be recorded every min at an intensity that evokes a response ⁇ 35% of the maximum evoked response. LTP will be induced using ⁇ -burst stimulation (4 pulses at 100Hz, with the bursts repeated at 5Hz and each tetanus including 3 ten-burst trains separated by 15sec).
  • mice For contextual and cued conditioning , mice will be placed in the conditioning chamber for 2min before the onset of a discrete tone (CS) (a 30s, 85dB sound at 2800Hz), as described [A17] . In the last 2s of the CS, mice will be given a 2s, 0.60mA foot shock (US) through the bars of the floor. After the CS/US pairing, mice will be left in the conditioning chamber for 30s and will then be placed back in their home cages. Freezing behavior will be scored using the Freezeview software (MED Ass). To evaluate contextual fear learning, freezing will be measured for 5min in the chamber in which the mice will be trained 24hr after training. To evaluate cued fear learning, 24hr after contextual testing, mice will be placed in a novel context for 2min (pre-CS test), after which they will be exposed to the CS for 3min (CS test), and freezing will be measured.
  • pre-CS test pre-CS test
  • CS test a novel context for 2min
  • CS test a novel
  • CBP levels will be measured with western blot using specific CBP antibodies.
  • the nuclear fraction will be contained in the pellet obtained from homogenated tissue, centrifuged at 7,700xg for 1min.
  • CBP HAT activity will be measured by immunoprecipitation from the lysis of hippocampal extracts using CBP antibodies. After isolation, HAT activity will be assessed using indirect enzyme-linked immunosorbent assay kit to detect acetyl residues according to the manufacturer's instruction (Upstate).
  • HDAC activity assay I will use a fluorimetric kit from Biovision (CA), according to the manufacturer instruction.
  • Western blot will be performed from snap-frozen in liquid nitrogen hippocampi. Nuclear proteins will be acid-extracted and separated onto a denaturing, 7%-12% acrylamide gel followed by electroblotting onto nitrocellulose. Acetylated histones (H3, H2A and H2B) will be detected using antibodies purchased from Upstate and the Amersham ECF Kit accordingly to the manufacturer protocol.
  • AD Alzheimer's disease
  • Hippocampal levels of acetylated histone 4 an acetylation important in memory formation [B19] were markedly reduced in an amyloid-depositing animal model after fear conditioning training. Without being bound by theory, changes in histone acetylation play an important role in AD.
  • Nicotinamide an HDAC III inhibitor
  • Nicotinamide was found to restore cognition in the triple transgenic mouse model of AD via a non-epigenetic mechanism involving reduction of cytosolic Thr231-phosphotau [B49].
  • histones H2B and 3 which are also known to play a key role in transcription and memory [B19, B25, B26], abnormally acetylated during memory processes in AD?
  • histone acetylation affected following A ⁇ elevation is histone acetylation affected following A ⁇ elevation?
  • histone acetyl-transferase CBP play a role in the reduction of histone acetylation in APP/PS1 mice and following A ⁇ elevation?
  • the therapies for AD include augmentation of the cholinergic system by usage of acetylcholinesterase inhibitors, or blockage of glutamate neurotoxicity through NMDA antagonists. These agents have a limited efficacy.
  • Major efforts are underway to inhibit tangle formation, to combat inflammation and oxidative damage, and to decrease A ⁇ load in the brain either by the use of agents that inhibit ⁇ and ⁇ secretases or increase ⁇ secretase, by the use of drugs that inhibit A ⁇ oligomerization, or by the use of treatments such as immunization with A ⁇ that appear to augment the removal of A ⁇ from the brain.
  • HDAC inhibition ameliorates deficits in hippocampal long-term potentiation in APP/PS1 mice.
  • TSA HDAC inhibitor
  • TSA was capable of rescuing the defect in LTP shown by slices from 3-4 month-old APP/PS1 mice (for a detailed description of the experiments showing the characterization of these mice [B50]).
  • the transgenic mice just start showing the synaptic plasticity and memory impairments [B14, B50]).
  • basal synaptic transmission (BST) was similar between APP/PS1 and WT mice.
  • HDAC inhibition is capable of rescuing the defect in LTP in the APP/PS1 animal model of A ⁇ deposition.
  • HDAC inhibition rescues the defect in associative memory by A ⁇ elevation.
  • the APP and PS1transgenes could affect neuronal function through different mechanisms [B23, B24], including direct effects by A ⁇ .
  • Full-length APP and its cleavage products could differently impact H4 acetylation.
  • a ⁇ per se is responsible for the deficit in H4 acetylation observed in our studies on transgenic mice.
  • APP/PS1 mice display a reduced endogenous level of histone 4 acetylation in response to a learning task.
  • HDAC inhibitors are known to acetylate other molecules besides histones [B49]
  • our next goal was to determine whether the effect of TSA on the defect in fear memory of APP/PS1 mice was linked, at least in part, to chromatin changes at the level of histone acetylation.
  • Acetylation of H4 was shown to play a key role in transcription and memory [B19].
  • CBP CRE-dependent gene expression
  • CREB transcription factor CREB
  • CBP coactivator-dependent gene expression
  • Histone acetylation induces chromosomal changes resulting in loss of chromosomal repression (see FIG. 23 ). This allows successful transcription of the underlying genes needed for synthesis of proteins underlying memory formation.
  • APP/PS1 mice display reduced CBP levels.
  • Western blot analysis from the hippocampus of 4 month old APP/PS1 mice revealed a significant decrease in CBP levels compared to WT controls ( FIG. 21 ) consistent with the observation that cerebral CBP levels are reduced in mice lacking functional PSs [B15].
  • AD is thought to begin as a synaptic disorder that progressively leads to greater neuronal dysfunction, leading to memory loss [B57].
  • HDAC inhibition counteracts the impairment of synaptic plasticity and memory following amyloid elevation.
  • NaB an additional and structurally dissimilar inhibitor
  • APP and PS1 transgenes could affect neuronal function through different mechanisms [B23, B24], we will determine whether A ⁇ per se is responsible for the deficit in histone acetylation observed in our studies on transgenic mice.
  • mice were divided into 4 groups: APP/PS1 with NaB, APP/PS1 with vehicle, WT with NaB and WT with vehicle.
  • Controls will be also performed using a latent inhibition training paradigm to exclude that HDAC inhibitors act through an effect on novel context alone or the electric shock instead of the association between them [B26].
  • animals will be pre-exposed to a novel context prior to receiving the electric shock so that the animal will form a spatial memory that blocks the formation of an associative contextual fear memory [B26].
  • TSA rescues the defect of contextual fear memory induced by a preparation containing oligomeric A ⁇ 42 see FIG. 24 .
  • TSA rescues the defect in LTP induced by 200 nM oligomeric A ⁇ 42 Since it has already been described that natural oligomers of human A ⁇ , in the absence of monomers and fibrils, markedly inhibit LTP in vivo [B29], we will apply 200 nM oligomeric A ⁇ 42 concurrently with TSA (1.65 ⁇ M) for 30 minutes to WT slices prior to inducing LTP. In interleaved control experiments we will apply oligomeric A ⁇ 42 alone, or TSA alone, or vehicle.
  • the method is a hybrid of the Morris water maze and the radial arm land maze.
  • the motivation for the animals is the immersion in water.
  • the mouse needs to swim in 6 alleys (arms) radiating from a central area until it finds a hidden (submerged) platform at the end of one of the arms.
  • the goal arm is kept constant for all trials, with a different start arm on successive trials, such that the learning criterion will be reached in 2 days. The number of incorrect arm entries will be.
  • the mice will perform visible platform testing to exclude the possibility that visual, motor and motivational deficits affect the outcome of the experiments.
  • mice will be injected with TSA or NaB, 2 hrs prior to performing the task. Vehicle will be used in control experiments. Similar experiments assessing reference memory will be also performed in A ⁇ 42 -infused mice (for these experiments mice will be divided into the following groups: A ⁇ 42 -infused animals + TSA or NaB or vehicle, vehicle-infused animals + TSA or NaB or vehicle).
  • HDAC inhibition rescues the deficits of LTP and memory. If not, as an alternative strategy, we will try higher concentrations of the HDAC inhibitors, or a structurally dissimilar HDAC inhibitor such as MS-275 which has also been shown to cross the blood-brain-barrier [B59]. Then, the rodent will undergo electrophysiological and behavioral testing as described herein. Without being bound by theory, oligomeric A ⁇ 42 will inhibits LTP and fear memory, and demonstrate that TSA ameliorates LTP and contextual fear memory following A ⁇ 42 treatment. TSA alone should not have any effect. Findings from young mice or A ⁇ -infused animals should be confirmed in 7 month old transgenics.
  • TSA and other HDAC inhibitors represent a new approach to AD treatment that appears to make the synapse more robust and resistant to the effects of A ⁇ .
  • HDAC inhibitors it has been criticized that inhibition of HDACs might alter gene expression globally and thus affect memory processes in a nonspecific manner.
  • Vecsey et al [B53] showed that TSA does not globally alter gene expression but instead increases the expression of specific genes during memory consolidation. They were able to show that HDAC inhibitors, including TSA, enhance memory and synaptic plasticity mainly by the activation of key genes that are dependent on CREB transcriptional activation [B53].
  • TSA may be capable of stopping memory degradation in the presence of A ⁇ accumulation as well as improving brain functions that have already deteriorated, as in the case of the 3-month-old APP/PS1 mouse.
  • HDAC inhibitors could be capable of reestablishing neural networks in the AD brain. This indicates that using small molecules to target HDACs in AD patients could facilitate access to long-term memories.
  • HDAC inhibitors with minimized side-effects are currently being developed by the pharmaceutical industry. It remains to be seen if these newer inhibitors can readily enter the brain and if they are as effective as TSA.
  • HDAC inhibitors could affect neuronal function through a variety of mechanisms including epigenetic and non-epigenetic changes [B19, B60].
  • the block of HDACs class I/II may increase the acetylation of non-histone substrates that, in turn, can contribute to the amplification of cellular processes associated with memory.
  • Green et al. [B49] showed that inhibition of class III NAD+-dependent HDACs using vitamin B3 restored cognitive deficits in the triple transgenic AD mice, via a mechanism involving the reduction of Thr231-phosphotau in the cytoplasm.
  • H4 acetylated histone 4
  • Example 6 Findings in Example 6 will be confirmed with the structurally dissimilar NaB.
  • NaB 1.2 g/Kg
  • controls will be performed on vehicle-infused mice and using a latent inhibition training paradigm, as well as cerebellum.
  • oligomeric A ⁇ 42 will affect H4 acetylation levels and oligomeric A ⁇ 42 will produce the same effects on acetylation levels of histones 2B and 3 as in transgenic mice. If not, we will try more prolonged applications of 200 nM oligomeric A ⁇ 42 through Alzet osmotic mini-pumps (1 day, 1 week, 1 month). Problems related to the use of a synthetic preparation containing A ⁇ 42 are greatly alleviated by the use of transgenic animals which produce natural forms of A ⁇ . Studies on H2B and 3 will provide a more complete picture of the type of epigenetic changes occurring at the level of histone acetylation. Finally, research in older mice will help understanding whether HDAC inhibitors might be used at older disease stages. Taken all together, the studies described herein will help establishing epigenetic changes as events occurring following A ⁇ elevation.
  • PTMs histone posttranslational modifications
  • H3 Lys4 methylation and H3 Lys56 acetylation were found to lead to gene expression.
  • histone modifications associated with the inactivation of gene transcription such as H3 Lys27 methylation and H2A Lys119 ubiquitination were found to cause gene silencing.
  • PTMs that are modified as a consequence of chronic neuronal exposure to oligomeric forms of A ⁇ 42 in mice. As described (see FIG.
  • oligomeric A ⁇ 42 will be infused into dorsal hippocampi of WT mice.
  • the hippocampi of A ⁇ 42-infused mice will be removed and compared to those of vehicle-infused mice.
  • mice will be sacrificed at 1 month (prior to plaque formation), 2 months (as plaques start to form), 3-4 months (at early stages of plaque formation), and 7 months (at late stages of plaque formation) of age to test when this histone PTMs occur.
  • epigenetic changes such as reduced histone acetylation, are likely to play an important role in the A ⁇ -induced damage of synaptic function and memory associated with AD.
  • CBP HAT activity is essential for enhancing transcription in vitro following PS1 stimulation. Furthermore, CBP levels in 3-4 month old APP/PS1 mice were found to be lower than in WT mice. In future experiments, we will extend this observation to older transgenic mice and following hippocampal A ⁇ infusion. In addition, we will determine if CBP HAT activity is affected both in young and older APP/PS1 mice as well as after A ⁇ infusion. Finally, we will determine whether stimulation of HAT activity through a recently synthesized HAT agonist, MOM, rescues the deficits in LTP, memory, and histone acetylation following A ⁇ elevation.
  • MOM recently synthesized HAT agonist
  • a HAT agonist such as N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxybenzamide (CTB or also referred to as compound 6J) [B61] was synthesized.
  • CTB N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxybenzamide
  • MOM benzamide HAT agonist
  • MOM (10 ⁇ M) or vehicle will be applied for 30 min prior to inducing LTP with the ⁇ -burst. WT littermate slices treated with MOM or vehicle will be used as controls.
  • MOM re-establishes normal reference memory.
  • cannulas will be implanted into the dorsal hippocampi of APP/PS 1 mice and WT littermates to deliver it directly into the hippocampi.
  • We will infuse 100 ⁇ g in 1 ⁇ l, slowly over 1 min. The infusion will occur 2 hrs prior to applying the foot shock for fear conditioning.
  • we will measure the amount of freezing at 24 hrs to assess contextual fear memory followed by cued fear memory at 48 hrs.
  • hippocampi and cerebella will be removed at 1 hr after training for fear conditioning and histone acetylation levels will be measured.
  • HAT agonists might ameliorate the defect in LTP, memory and histone acetylation following overexpression of APP and PS1 transgenes.
  • the protocol will also be carried out for testing the effect of the HAT activator, CTB.
  • HDAC activity through a new fluorimetric assay on hippocampi using the experimental paradigm as in the HAT assay (basal, 1 min, 5 min, 20 min and 1 hour after foot shock).
  • HAT assay basic, 1 min, 5 min, 20 min and 1 hour after foot shock.
  • HATs are involved in the reduction of histone acetylation. These include GNATs family, MYST family, p300, and ACTR/SRC-1. We will measure levels and activity of these HATs.
  • Double transgenic mice will be obtained by crossing APP and PS1 animals (genotyped by PCR) [B20, B21, B50].
  • APP and PS1 animals geneotyped by PCR
  • For A ⁇ experiments we will use C57B16 mice which will be obtained from a breeding colony. All the mice will be maintained on a 12 h light/dark cycle (with lights on at 6:00 A.M.) in temperature- and humidity-controlled rooms. Food and water will be available ad libitum.
  • Electrophysiological studies We will cut 400 ⁇ m hippocampal slices from C57B16 mice and maintain them in an interface chamber at 29° C for 90 min prior to recording, as previously reported [B11].
  • the bath solution will consist of 124.0 mM NaCl, 4.4 mM KC1, 1.0 mM Na 2 HPO4, 25.0 mM NaHCO 3 , 2.0 mM CaCl 2 , 2.0 mM MgSO 4 , and 10.0 mM glucose, continuously bubbled with 95% O 2 and 5% CO 2 .
  • a stimulating electrode we will use a bipolar tungsten electrode, placed at the level of the Schaeffer collateral fibers.
  • Contextual fear conditioning will be assessed as previously described [B14, B21]. Mice will be placed in a conditioning chamber for 2 min before the onset of a tone (CS) (a 30 s, 85 dB sound at 2800 Hz). In the last 2 s of the CS, mice will be given a 2 s, 0.45 mA foot shock (US) through the bars of the floor. Then, the mice will be left in the conditioning chamber for another 30 s. Freezing behavior, defined as the absence of movement except for that needed for breathing, will be scored using the Freezeview software. Contextual fear learning, a type of memory for which hippocampal function is indispensable, will be evaluated 24 hrs after training by measuring freezing for 5 min in the chamber in which the mice will be trained.
  • CS tone
  • US foot shock
  • Cued fear learning a type of memory that depends upon amygdala function, will be evaluated 24 hrs after contextual testing by placing mice in a novel context for 2 min (pre-CS test), after which they will be exposed to the CS for 3min (CS test), and freezing will be measured.
  • Sensory perception of the shock will be determined through threshold assessment. Briefly, the electric current (0.1 mA for 1 s) will be increased at 30 s intervals by 0.1 mA to 0.7 mA. Threshold to flinching (first visible response to shock), jumping (first extreme motor response), and screaming (first vocalized distress) will be quantified for each animal by averaging of the shock intensity at which each animal manifest a behavioral response of that type to the foot shock. No difference in the sensory threshold assessment should be observed among different groups of mice in experiments in which fear conditioning is tested if the experimental procedure does not affect the sensory threshold of the animals.
  • mice will be checked with the open-field test.
  • the open field will be an arena made of white acrylic with internal dimensions of 72 X 72 X 33 cm (An area measuring 36 X 36 cm in the centre of the open field will be defined as the 'central zone').
  • Mice will be placed in the center of a standard open field and their behavior monitored for 1 hr and scored for proportion of time in the center compartment vs. periphery, and number of entries into the center compartment. Mice will be returned for a second hour block after 24 hr. No difference in exploratory behavior as demonstrated by a similar percentage of time spent in the center compartment and the number of entries into the center compartment should be observed if the manipulation does not affect exploratory capabilities of the mice.
  • the first day of the protocol will be a training day. Mice will be trained to identify the platform location by alternating between a visible and a hidden platform in a goal arm. The final 3 trials on day 1 and all 15 trials on day 2 will use a hidden escape platform to force mice to use spatial cues to identify the location of the goal arm. To avoid learning limitations imposed by exhausting practice and to avoid fatigue that may result from consecutive trials, spaced practice training will be established by running the mice in cohorts of 4 and alternating different cohorts through the 15 training trials over 3 hours testing periods each day. On day 1, a visible platform will be placed in a goal location.
  • Mouse 1 of cohort 1 will be gently placed in the pool near the perimeter of the wall of the first start arm (specified on a score sheet) and facing the center of the pool. The number of incorrect arm entries (entries in arms with no platform) will be counted. If the animal enters the incorrect arm it is gently pulled back to the start arm. Each trial will last up to 1 minute. Failure to select an arm after 15 seconds will be counted as an error and the mouse will be returned to the start arm. After 1 minute, if the platform has not been located, the mouse will be guided gently through the water by placing a hand behind it to direct it towards the platform. The mouse will rest on the platform for 15 seconds. After completing the trial, the mouse will be removed from the pool, gently towel dried and placed back into its cage under a heat lamp.
  • mice from the first cohort After all the mice in the first cohort have had a trial to locate a visible platform, the platform will be switched from visible to hidden. After each mouse from cohort 1 completes six alternating trials between visible and hidden platforms, the mice will be left to rest under a heating source, and mice from the second cohort will be tested in the same way. After completing the six alternating trials, mice from cohort 2 will return to their cages to rest. Next, mice from the first cohort will complete trials 7-12 again using the alternating visible-hidden platform location. During resting time for mice from the first cohort, mice from the second cohort will complete trials 7-12. At this point, all mice will have to perform 3 hidden platform trials.
  • the cannulas will be fixed to the skull with acrylic dental cement (Paladur).
  • mice For Morris water maze, mice will be injected 20 min prior to performing each session and the probe trial, whereas for fear conditioning mice will receive a single injection 20 min before the training. Mice will be handled once a day for 3 days before behavioral experiments. During infusion animals will be handled gently to minimize stress. After infusion, the needle will be left in place for another minute to allow diffusion. After behavioural testing, a solution of 4% methylene blue will be infused into the cannulas. Animals will be sacrificed and their brains removed, frozen, and then cut at -20° with cryostat for histological localization of infusion cannulas.
  • Oligomeric A ⁇ 42 will be prepared from commercially available synthetic peptides (American Peptides Co), as described [B63, B64]. Briefly, the lyophilized peptide will be resuspended in cold 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Sigma) and aliquoted in polypropylene vials. After 24 hrs the HFIP solution will be allowed to evaporate in a fume hood until a thin film of peptide is formed on the bottom of the vials. Peptide films will be dried under gentle vacuum and stored in sealed vials at -20°C.
  • HFIP 1,1,1,3,3,3-hexafluoro-2-propanol
  • anhydrous DMSO (Sigma) will be added to obtain a pure monomeric A ⁇ /DMSO solution that will be sonicated for 10 min [B63].
  • Oligomeric A ⁇ 42 will be obtained by incubating an aliquot of monomeric A ⁇ /DMSO solution in sterile PBS at 4°C overnight.
  • the quality of these A ⁇ preparations will be routinely controlled using Western blot analysis in which A ⁇ samples will be resolved by Tris-Tricine PAGE under non-denaturing/nonreducing conditions, and then transferred on nitrocellulose membrane. Subsequent Western blotting will be carried out after membrane incubation with the anti-human A ⁇ monoclonal antibody 6E10 (Signet Lab). The immunostaining will be revealed by horseradish peroxidase chemi-luminescence.
  • Histone acetylation assay Western blot will be performed from snap-frozen in liquid nitrogen hippocampi and cerebella. Tissue will be homogenized in lysis buffer (62.5 mM Tris-HCl pH 6.8, 3% SDS, 1 mM DTT) and incubated at 4 °C for 10 min, then sonicated before centrifugation at 2,000 rpm for 5 min. Whole cell extracts will be electrophoresed on 10-20% gradient PAGE gel (Invitrogen) and then immunoblotted. Antibodies will be used at a 1:1,000 concentration for immunoblotting. All anti-histone antibodies will be purchased from Millipore. ⁇ -III-Tubulin antibody will be purchased from Promega.
  • Immunoblot data will be quantified by measuring the band intensity using imaging software (NIH ImageJ). For quantitative immunoblot analysis, equal amounts of proteins will be loaded into each lane. To confirm equal loading, blots will be reprobed with corresponding pan-antibodies or antibodies for house-keeping proteins such as ⁇ -III-Tubulin. For quantification, we always use a signal in the linear range.
  • Immunoprecipitated histones will be purified by reverse-phase HPLC in the PSR or by SDS-PAGE, then subjected to enzymatic digestion. Resulting peptides will be analyzed by LC-MS/MS on a Waters Qtof mass spectrometer equipped with a Dionex nanflow LC. The standard digestion protocol using trypsin is not feasible due to the number of Lys residues in the N-terminal portion of histones, resulting in peptides too small to be analyzed.
  • CBP levels will be measured with western blot using specific CBP antibodies.
  • the nuclear fraction will be contained in the pellet obtained from homogenated tissue, centrifuged at 7,700xg for 1min.
  • CBP HAT activity will be measured by immunoprecipitation from the lysis of hippocampal extracts using CBP antibodies. After isolation, HAT activity will be assessed using indirect enzyme-linked immunosorbent assay kit to detect acetyl residues according to the manufacturer's instruction (Upstate).
  • HDAC activity will be measured using a fluorimetric kit from Biovision (CA), according to the manufacturer instruction.
  • Example 7 Cell Viability Assays for ACHN, U251, NCI-ADR-RES, A549, Hs578T, CCRF-CEM
  • Cells and culture medium All cell lines were purchased from the ATCC and were expanded and archived under liquid nitrogen at CDAS as low passage aliquots. Cells were maintained and passaged in recommended and optimal culture medium (ACHN: EMEM, 2 mM L-Gln, 10% FBS; A549: Ham's F12, 10% FBS; U251: RPMI 1640,2 mM L-Gln, 10% FBS; Hs578T: DMEM, 4 mM L-Gln, 1 U/mL of Bovine Insulin, 10% FBS; CCRF-CEM: ATCC RPMI, 2 mM L-Gln, 10% FBS; NCI-ADR-RES: RPMI 1640, 2 mM L-Gln, 10% FBS). All experiments were carried out with cells which had undergone less than 20 passages. Optimal seed densities were determined for all cell lines. All cells were plated at 1500 cells per well except CCRF-CEM which was plated at 6000 cells per
  • YF2 was supplied as a 80 mM stock solution in 100% DMSO.
  • Vinblastine was purchased from Sigma (Catalogue Number V-1377) and resuspended at 1 X 10 -2 M in 100% DMSO. All dilutions for both drugs were carried out in culture medium containing 0.2% DMSO such that the final solvent concentration never exceeded 0.1 %.
  • Drug treatment YF2 tested at 10 concentrations (0.03, 0.1, 0.25, 0.5, 1,2.5, 5, 15,40 and 80 ⁇ M) in triplicate wells. Vinblastine was used as a reference control and tested at 10 concentrations in a half-log series (0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3,1,3, and 10 ⁇ M). Cells were resuspended in medium at the appropriate concentration and 180 ⁇ l (1500 or 6000 cells) was added to each well following which 20 ⁇ l of drug at 10x of the final concentration was added to attain the desired drug concentration in every well. The drug treatment plates were incubated at 37°C for 72 hours, following which cell viability was assayed by the Cell Titer Glo or Cyquant method as described below.
  • Cell Titer Glo Assay Following the 72 hour drug treatment period, the assay plates were centrifuged, and 100 ⁇ L of the medium was aspirated and replaced with 100 J.1L of Cell Titer Glo reagent (Promega) according to the manufacturer's recommended protocol. The reagent was mixed with the cells and the luminescence measured using a Perkin Elmer Envision instrument. The average luminescence signal obtained from wells containing untreated cells which had been incubated for the entire length of the assay period was used to set the 100% viability value. The percent proliferation was calculated as (Test signal)/(Avg. plate background signal) x 100. The % viability was graphed against drug concentration to calculate an IC 50 for each drug.
  • Cyquant Assay Following the 72 hour drug treatment period the assay plates were centrifuged, the medium discarded, and frozen overnight. The plates were assayed using the Cyquant TM reagent (Invitrogen) according to the manufacturer's recommended protocol. The average fluorescence signal obtained from wells containing untreated cells which had been incubated for the entire length of the assay period was used to set the 100% proliferation value. The percent proliferation was calculated as (Test signal)/(Avg. plate background signal) x 100. The % proliferation was graphed against drug concentration to calculate an IC 50 for each drug.
  • Example 8 YF2 Increases Histone Acetylation by HAT Activation, Not HDAC Inhibition
  • HDAC inhibition causes an increase in histone acetylation.
  • the inventors examined whether histone acetylation occurred via HDAC inhibition.
  • Assay Conditions A series of dilution of the test compounds were prepared with 10% DMSO in assay buffer and 5 ⁇ l of the dilution was added to a 50 ⁇ l reaction so that the final concentration of DMSO is 1% in all of reactions. All of the enzymatic reactions were conducted in duplicate at 37°C for 30 minutes except of HDAC11 at room temperature for 3 hours.
  • the 50 ⁇ l reaction mixture contains HDAC assay buffer, 5 ⁇ g BSA, an HDAC substrate, an HDAC enzyme and a test compound. After enzymatic reactions, 50 ⁇ l of HDAC Developer was added to each well and the plate was incubated at room temperature for an additional 20 minutes. Fluorescence intensity was measured at an excitation of 360 nm and an emission of 460 nm using a Tecan Infinite M1000 microplate reader.
  • HDAC activity assays were performed in duplicates at each concentration.
  • the fluorescent intensity data were analyzed using the computer software, Graphpad Prism.
  • the fluorescent intensity (F t ) in each data set was defined as 100% activity.
  • the fluorescent intensity (F b ) in each data set was defined as 0% activity.
  • Compound OA2 has fluorescence at assay condition; therefore the fluorescent intensity at different concentration of OA2 was defined as background (Fb).
  • the IC 50 value was determined by the concentration causing a half-maximal percent activity.
  • FIG. 52 corresponds to the results shown in Table 4.
  • Table 5 HDAC3/NCOR2 Assay - Data for the Effect of OA2 on HDAC3/NCOR2 Activity OA2 (Log [nM]) HDAC Activity (Fluorescence count) Background (Fluorescence count) % Activity Repeat1 Repeat2 Repeat1 Repeat2 Repeat1 Repeat2 No CPD 10787 10452 805 828 101.71 98.29 0.5 10928 9694 813 976 102.35 89.76 1.0 10423 10379 812 818 98.01 97.56 1.5 10752 10231 813 803 101.44 96.12 2.0 10827 10078 809 798 102.25 94.61 2.5 10718 10173 818 803 101.07 95.51 3.0 10587 10073 831 811 99.62 94.38 3.5 10362 10080 854 824 97.14 94.27 4.0 11530 10216 927 898 108.31 94.90
  • FIG. 53 corresponds to the results shown in Table 5.
  • Table 6 HDAC5FL Assay - Data for the Effect of OA2 on HDAC5FL Activity OA2 (Log [nM]) HDAC Activity (Fluorescence count) Background (Fluorescence count) % Activity Repeat 1 Repeat2 Repeat 1 Repeat2 Repeat 1 Repeat2 No CPD 4492 4892 345 348 95.40 104.60 0.5 4686 4386 355 343 99.80 92.90 1.0 4802 4581 341 347 102.59 97.50 1.5 4835 4874 359 342 103.20 104.10 2.0 5071 4991 344 356 108.64 106.80 2.5 5068 5006 344 108.60 107.17 3.0 4944 4685 342 354 105.76 99.80 3.5 4773 4686 353 101.30 99.30 4.0 4987 4983 449 407 104.91 104.82 4.5 4570 4514 451 398 95.40 94.11 5.3
  • FIG. 54 corresponds to the results shown in Table 6.
  • Table 7. HDAC7 Assay - Data for the Effect of OA2 on HDAC7 Activity OA2 (Log [nM]) HDAC Activity (Fluorescence count) Background (Fluorescence count) % Activity Repeat 1 Repeat2 Repeat 1 Repeat2 Repeat 1 Repeat2 No CPD 7528 7176 382 377 102.52 97.48 0.5 7578 7200 394 383 103.11 97.69 1.0 6756 6763 385 386 91.37 91.47 1.5 7471 7705 389 381 101.63 104.98 2.0 7679 7196 390 380 104.61 97.68 2.5 7071 7068 385 398 95.80 95.75 3.0 7083 7269 384 392 96.02 98.69 3.5 7453 6898 397 462 100.73 92.77 4.0 6801 7568 416 534 90.73 101.73 4.5 7238 7518 554 565 95
  • FIG. 55 corresponds to the results shown in Table 7.
  • Table 8. HDAC8 Assay - Data for the Effect of OA2 on HDAC8 Activity OA2 (Log [nM]) HDAC Activity (Fluorescence count) Background (Fluorescence count) % Activity Repeat1 Repeat2 Repeat1 Repeat2 Repeat1 Repeat2 No CPD 3492 3483 346 346 100.14 99.86 0.5 3541 3581 339 342 101.88 103.15 1.0 3519 3391 349 342 101.02 96.94 1.5 3539 3456 336 331 102.04 99.40 2.0 3757 3425 338 340 108.80 98.23 2.5 3451 3428 335 341 99.09 98.36 3.0 3398 2995 337 347 97.28 84.45 3.5 3808 3407 346 366 109.88 97.12 4.0 3361 3365 433 374 94.14 94.27 4.5 3045 3090 375 364 85.17 86.60 5.3
  • FIG. 56 corresponds to the results shown in Table 8.
  • Table 9. HDAC10 Assay - Data for the Effect of OA2 on HDAC10 Activity OA2 (Log [nM]) HDAC Activity (Fluorescence count) Background (Fluorescence count) % Activity Repeat 1 Repeat2 Repeat 1 Repeat2 Repeat 1 Repeat2 No CPD 11695 12141 497 507 98.05 101.95 0.5 10894 12032 492 501 91.08 101.05 1.0 12341 12402 497 492 103.77 104.31 1.5 12564 12368 525 500 105.57 103.85 2.0 12262 12573 500 497 103.04 105.77 2.5 12472 12556 500 493 104.90 105.64 3.0 11935 12471 530 521 99.94 104.64 3.5 11622 12684 501 607 96.95 106.25 4.0 11588 12318 597 547 96.50 102.89 4.5
  • FIG. 57 corresponds to the results shown in Table 9.
  • Table 10 HDAC11 Assay - Data for the Effect of OA2 on HDAC11 Activity OA2 (Log [nM]) HDAC Activity (Fluorescence count) Background (Fluorescence count) % Activity Repeat 1 Repeat2 Repeat 1 Repeat2 Repeat 1 Repeat2 No CPD 2840 2860 426 406 99.59 100.41 0.5 2761 2530 411 423 96.30 86.81 1.0 2828 2898 425 415 98.93 101.81 1.5 2765 2851 411 406 96.82 100.35 2.0 2812 2864 408 409 98.75 100.88 2.5 2672 2655 412 408 92.93 92.24 3.0 2829 2806 417 424 98.95 98.01 3.5 2719 2712 427 463 93.43 93.14 4.0 2835 2860 467 524 96.12 97.14 4.5 3289 3064 699 617 108.09 98.
  • FIG. 58 corresponds to the results shown in Table 10.
  • Table 11 Sirtuin 1 Assay - Data for the Effect of OA2 on Sirtuin 1 Activity OA2 (Log [nM]) HDAC Activity (Fluorescence count) Background (Fluorescence count) % Activity Repeat 1 Repeat2 Repeat 1 Repeat2 Repeat 1 Repeat2 No CPD 5823 5974 412 410 97.91 100.64 0.5 5627 5940 414 420 94.26 99.92 1.0 5240 5913 422 413 87.25 99.42 1.5 5980 5273 418 457 100.27 87.48 2.0 5827 5527 411 411 97.98 92.56 2.5 6028 5987 413 416 101.56 100.81 3.0 6454 5681 422 452 108.86 94.87 3.5 5782 5964 422 426 96.93 100.23 4.0 5786 5408 442 441 96.69 89.85 4.5 5976
  • FIG. 59 corresponds to the results shown in Table 11.
  • Table 12. Sirtuin 2 Assay - Data for the Effect of OA2 on Sirtuin 2 Activity OA2 (Log [nM]) HDAC Activity (Fluorescence count) Background (Fluorescence count) % Activity Repeat 1 Repeat2 Repeat1 Repeat2 Repeat2 No CPD 3910 3919 413 419 99.87 100.13 0.5 3835 3981 420 413 97.71 101.89 1,0 3780 3821 406 422 96.21 97.38 1.5 3858 3954 408 410 98.59 101.33 2.0 3712 3912 420 413 94.20 99.91 2.5 3729 3788 409 420 94.74 96.43 3.0 3714 3861 405 409 94.53 98.73 3.5 3806 3856 422 417 96.80 98.23 4.0 3844 3883 425 426 97.71 98.83 4.5 3717 3811 485 480 92.45 95
  • FIG. 60 corresponds to the results shown in Table 12.
  • Table 13 HDAC6 Assay - Data for the Effect of OA2 on HDAC6 Activity OA2 (Log [nM]) HDAC Activity (Fluorescence count) Background (Fluorescence count) % Activity Repeat 1 Repeat2 Repeat 1 Repeat2 Repeat 1 Repeat2 No CPD 5844 5616 773 733 102.29 97.71 0.5 5998 6074 832 737 104.75 106.28 1.0 6006 5728 747 704 106.10 100.51 1.5 5541 6126 746 706 96.75 108.50 2.0 5733 5981 748 731 100.33 105.31 2.5 5678 5677 763 709 99.30 99.28 3.0 5717 5446 758 716 100.06 94.62 3.5 5575 5616 781 735 96.79 97.61 4.0 5516 5789 828 786 94.62 100.10 4.5 4994 5418 1081 1030 79
  • FIG. 74 corresponds to the results shown in Table 13.
  • SAHA is an HDAC inhibitor (HDACi). It serves as a positive control for HDACs.
  • FIGS. 61-63 show the inhibitory effect of SAHA on the HDACs HDAC1, HDAC3/NCOR2, and HDAC6. SAHA also inhibited HDAC5FL, HDAC7, HDAC8, HDAC10, Sirtuin 1, and Sirtuin 2 (scc Table 1).
  • AD Alzheimer's disease
  • AP ⁇ -amyloid
  • LTP long-term potentiation
  • memory is modulated by epigenetics through regulation of gene expression, deregulation of one of the epigenetic mechanisms such as histone (H) acetylation, might lead to memory disruption. Reduction of histone acetylation causes the chromatin structure to close, so that the information contained within the DNA might be less amenable to transcription factors and memory formation [9] .
  • HDAC inhibitors The main strategy that is currently used to up-regulate histone acetylation involves HDAC inhibitors.
  • the pleiotropic effect of nonspecific HDAC inhibition may hamper their therapeutic potential [10-13] .
  • CBP and PCAF hippocampal levels of two HATs, CBP and PCAF, are reduced following A ⁇ elevation.
  • HAT activator YF2.
  • HDAC inhibitors have been shown to enhance LTP and contextual fear memory, a form of associative memory in which animals must associate a neutral stimulus with an aversive one [P17] . Also, memory and LTP deficits of CBP +/- mice were reversed by HDAC inhibition [P15] .
  • HDAC inhibition The potential of inhibiting HDACs to counteract neurodegenerative disorders has been widely explored [14] . For instance, in a set of experiments, Tsai et al.
  • HDAC inhibitors induced sprouting of dendrites, increased number of synapses, and reinstated learning and access to long-term memories in the CK-p25 Tg mouse model of neurodegeneration [15,16] .
  • HDAC inhibitor TSA ameliorates LTP and contextual fear conditioning (FC) in the double Tg APP(K670M:N671L)/PS1(M146L, line 6.2) (APP/PS1) mouse model of amyloid deposition [17] .
  • FC LTP and contextual fear conditioning
  • HATs can be divided in two main groups, the nuclear HATs and cytoplasmic HATs [18] .
  • Nuclear A-type HATs can be grouped into at least 4 different families based on sequence conservation within the HAT domain: Gcn5 and p300/CBP associated factor (PCAF), MYST (MOZ, Ybf2/ Sas3, Sas2 and Tip60), p300 and CBP (named for the two human paralogs p300 and CBP) and Rtt109. While the Gcn5/PCAF and MYST families have homologs from yeast to man, p300/CBP is metazoan-specific, and Rtt109 is fungal-specific.
  • PCAF Gcn5 and p300/CBP associated factor
  • MYST MOZ, Ybf2/ Sas3, Sas2 and Tip60
  • p300 and CBP named for the two human paralogs p300 and CBP
  • Rtt109 is fungal-specific.
  • Cytoplasmic B-type HATs such as HAT1 are involved in histone deposition [P22] .
  • Marmorstein and Roth 2001, Curr Opin in Genet and Develop., 11:155-161 ) list in Table 1 the HAT families and their transcriptional-related functions, the reference which is incorporated by reference in its entirety.
  • HAT families have been described, such as the steroid receptor coactivators, TAF250, ATF-2, and CLOCK, their HAT activities have not been investigated as extensively as the major HAT classes [18] . These 4 families show high sequence similarity within families but poor to no sequence similarity between families. Furthermore, the size of the HAT domain of the different families is different [P22] . Interestingly, HATs are highly conserved in mammals [P22] . Of all these HATs, three were shown to be involved in memory: CBP, p300 [19,20] , and PCAF [21] . Interestingly, both CBP and PCAF levels are reduced by A ⁇ elevation.
  • HAT activators are a viable approach to enhance histone acetylation.
  • Two scaffolds for HAT activators have been identified. The first one includes CTPB and its derivative CTB [22,23] . The second one includes only one compound, nemorosone [24] .
  • CTPB/CTB were found to be insoluble and membrane-impermeable [22,23] .
  • CTPB has unfavorable characteristics to be used in CNS diseases (MW equal to 553,29, clogP equal to 12.70) and the clogP of CTB is 5.13.
  • Nemorosone has a MW of 502 and a clogP of 8.42.
  • AD therapies have limited efficacy. Major efforts are underway to inhibit tangle formation, to combat inflammation and oxidative damage, and to decrease A ⁇ load in the brain [26-28] .
  • APP, A ⁇ , and the secretases in normal physiological function [29-31] might present a problem in providing effective and safe approaches to AD therapy.
  • Developing agents that interact with A ⁇ targets that lead to neuronal dysfunction is another approach that is currently tested by many laboratories.
  • HAT activators represent a new class of compounds that might effectively counteract the disease progression.
  • CBP CREB binding protein
  • PCAF p300/CBP associated factor
  • HATs histone acetyltransferases
  • YF2 HAT Activator compound
  • YF2 for CBP, PCAF, and GCN5 are 2.75 ⁇ M, 29.04 ⁇ M and 49.31 ⁇ M, respectively. Additionally, YF2 did not interfere with p300 and HDAC activity (HDAC 1, 3, 5, 6, 7, 8, 10, 11, and sirt1-2). YF2 also increases p300 activity as shown in FIG. 72 .
  • the amount of YF2 in the brain was higher than that in the plasma with an AUC 0-t ratio of 8.2 and 10.8 for i.p. and i.v. administration, respectively.
  • the elimination half-lives of YF2 in the brain and plasma were ⁇ 40 min.
  • the T max values in the brain and plasma were similar, indicating that the distribution of YF2 to the brain is also fast. Additionally, in acute toxicity experiments YF2 did not induce any adverse effects up to 300mg/kg (i.p.).
  • YF2 increases histone acetylation in mouse hippocampus.
  • the compound was i.p. administered at 20 mg/Kg, mice were sacrificed 30 min later, and hippocampi were removed and quickly frozen for WB analysis.
  • YF2 increased acetylation of histone lysines that were shown to be involved in memory formation (H3K4, H3K9, H3K14, H4K5, H4K8, H4K12, H4K16, and H2B) [P15, P33] .
  • mice have been well characterized with respect to AD pathology [P34-38] . They start to show synaptic and memory impairment as early as 3 months of age [P34] .
  • Transgenic mice with a double mutation APP (K670M:N671L)/ PS1(M146L) (line 6.2) start to develop large plaques in cortex and hippocampus at the age of 8-10 weeks. They have a reduction in LTP by 3 months of age together with impairment of contextual fear memory and spatial working memory.
  • Histone 3 and 4 acetylation is decreased following A ⁇ 42 elevation.
  • HATs levels CBP and PCAF
  • HDAC inhibition is beneficial against damage of synaptic function and memory following A ⁇ 42 elevation.
  • HAT activation is beneficial against memory loss following A ⁇ 42 elevation.
  • Example 11 HAT ACTIVATORS WITH HIGH AFFINITY AND GOOD SELECTIVITY FOR CBP AND/OR PCAF AND/OR P300
  • HAT Assay kit from Active Motif (USA, CA).
  • HAT Assay kit from Active Motif (USA, CA).
  • the catalytic domains for the remaining HATs will be produced using New England Biolabs K. lactis Protein Expression Kit.
  • the candidate compounds In addition to being potent activators of CBP and/or PCAF (EC 50 ⁇ 100nM), the candidate compounds must also be selective. When assayed against all other HATs, they must show at least a 50 fold greater potency towards CBP and/or PCAF.
  • blood samples will be obtained from test animals following a single acute administration (collected at 5min, 15min, 30 min, 1hr, 2hrs, 4hrs, and 24hrs). Blood will be harvested by retro-orbital puncture, collected in heparanized tubes, and plasma obtained by centrifugation. Samples will be analyzed by LC-MS to measure the amounts of the candidate compound and possible metabolites. An indication of brain uptake and BBB penetration will be obtained by tissue extraction of the candidate compound from brain. Briefly, brain homogenates will be centrifuged 11,000rpm for 10 min.
  • brain uptake reflects concentration of the blood.
  • a peak brain/blood concentration ratio >1 will indicate that brain uptake for our compound is comparable with that of known CNS drugs in clinical use.
  • the brain/blood ratio for minaprine, a 6-phenylaminopyridazine CNS drug is >2 [P48] .
  • Synaptic dysfunction is a major hallmark of AD [P50] .
  • a qualifying aspect of our drug screening protocol will include a measurement of the effect of the newly-synthesized compounds onto synaptic function.
  • the APP/PS1 mouse presents an impairment of LTP by the age of 3 months [P34] and therefore permits a relatively fast assessment of synaptic function without waiting a long time for mice aging.
  • LTP because it is a type of synaptic plasticity that is thought to underlie learning and memory.
  • YF2 rescues the A ⁇ -induced reduction of LTP we will screen the compounds indicated by our MedChem studies to select those that can re-establish normal LTP. The compounds will be applied for 30 min using the same experimental protocol as in FIG. 66A.
  • Controls will be performed on slices from APP/PS1 mice treated with vehicle, and WT mice treated with compound or vehicle. If the compounds re-establish normal LTP in APP/PS1 slices, we will conclude that the compounds can rescue impairment of synaptic plasticity in APP/PS1 mice. We will also investigate another important aspect of the disease, the cognitive impairment (see Example 14).
  • mice will be obtained by crossing APP(K670M:N671L) with PS1(M146L) (line 6.2) animals.
  • the genotype will be identified by PCR on tail samples [P51-P53] .
  • Electrophysiology will be performed on males (see description in Gong et al [P54] ).
  • Example 14 FURTHER SCREENING OF HAT ACTIVATORS TO EXAMINE IF THEY AMELIORATE COGNITIVE ABNORMALITIES IN APP/PS1 MICE
  • treatment with a novel HAT activator indicated by Example 13 can rescue the cognitive deficits in 3 and 6 month old APP/PS1 mice.
  • the treatment will be performed with the same timing (i.e. 30min before training for fear conditioning or before the 1 st and 2 nd group of tests for the RAWM).
  • Conditions to be tested include: APP/PS1 and WT treated with HAT activators, APP/PS1 and WT treated with vehicle.
  • mice After behavioral testing mice will be sacrificed and their blood and brains used for A ⁇ level, Tau protein, TARDBP and TDB levels, and alpha-synuclein measurements.
  • we will measure hippocampal acetyl-H4 levels after administration of the compounds 30 min prior to training for fear conditioning and removal of the hippocampi 1hr after the electric shock (APP/PS1 mice have been shown to have a reduction of acetylated H4 after the electric shock [P21] ).
  • APP/PS1 mice have been shown to have a reduction of acetylated H4 after the electric shock [P21] ).
  • we will screen them with a battery of assays focusing on two areas that have resulted in the withdrawal of many drugs from the market: drug-drug interactions, hERG channel blockage (see Example 12).
  • Histone acetylation assay Western blot will be performed from snap-frozen in liquid nitrogen hippocampi. Tissue will be homogenized in RIPA buffer, then sonicated before centrifugation at 10,000 rpm for 5 min. Whole cell extracts will be electrophoresed on 10-20% gradient PAGE gel (Invitrogen) and then immunoblotted. Antibodies will be used at a 1:1,000 concentration for immunoblotting. All anti-histone antibodies will be purchased from Millipore. Immunoblot data will be quantified by measuring the band intensity using imaging software (NIH ImageJ).
  • a ⁇ levels will be performed on homogenates of frozen hemi-brains and plasma as previously described [P34] .
  • alpha-synuclein levels will be performed on homogenates of frozen hemi-brains using an ⁇ -Synuclein ELISA Kit (Catalog # NS400; Millipore, Billerica, MA) according to manufacturer's instructions.
  • TARDBP/TDP-43 levels will be performed on homogenates of frozen hemi-brains using a Human TAR DNA binding protein 43, TARDBP/TDP-43 ELISA Kit (Catalog # E1951h; Wuhan EIAab Science Co, Wuhan, China) according to manufacturer's instructions.
  • Determination of total Tau and phosphorylated Tau (Thr 231) levels will be performed on homogenates of frozen hemi-brains and plasma using assay and kits according to manufacturer's instructions available from MesoScale Discovery (Gaithersburg, MD) (see http://www.mesoscale.com/catalogsystemweb/webroot/products/assays/alzheimers.aspx).
  • mice will be performed in blind. Results will be expressed as Standard Error Mean (SEM). Level of significance will be set for p ⁇ 0.05. Results will be analyzed with ANOVA with post-hoc correction with drug or genotype as main effect.
  • Example 15 FURTHER SCREENING OF HAT ACTIVATORS TO EXAMINE IF THEY AMELIORATE COGNITIVE ABNORMALITIES IN MOUSE MODELS FOR HUNTINGTON'S DISEASE
  • Example 13 We will examine whether treatment with a HAT activator compound indicated by Example 13 can rescue the cognitive deficits in a mouse model of Huntington's Disease (e.g., FVB-Tg(YAC128)53Hay/J and FVB/NJ-Tg(YAC72)2511Hay/J mice, available from the Jackson Laboratory, Bar Harbor ME).
  • Huntington's Disease e.g., FVB-Tg(YAC128)53Hay/J and FVB/NJ-Tg(YAC72)2511Hay/J mice, available from the Jackson Laboratory, Bar Harbor ME.
  • RAWM and contextual FC two types of tests assessing different types of memory (reference ad associative). The treatment will be performed with the same timing (i.e. 30min before training for fear conditioning or before the 1 st and 2 nd group of tests for the RAWM).
  • Conditions to be tested include: Huntington's Disease mice and WT treated with HAT activators, Huntington's Disease mice and WT treated with vehicle. After behavioral testing mice will be sacrificed and their blood and brains used for Huntingtin protein level measurement. As a control for effectiveness of HAT activation, we will measure hippocampal acetyl-H4 levels after administration of the compounds 30 min prior to training for fear conditioning and removal of the hippocampi 1hr after the electric shock. Finally, we will screen them with a battery of assays focusing on two areas that have resulted in the withdrawal of many drugs from the market: drug-drug interactions, hERG channel blockage (see Example 12).
  • Histone acetylation assay Western blot will be performed from snap-frozen in liquid nitrogen hippocampi. Tissue will be homogenized in RIPA buffer, then sonicated before centrifugation at 10,000 rpm for 5 min. Whole cell extracts will be electrophoresed on 10-20% gradient PAGE gel (Invitrogen) and then immunoblotted. Antibodies will be used at a 1:1,000 concentration for immunoblotting. All anti-histone antibodies will be purchased from Millipore. Immunoblot data will be quantified by measuring the band intensity using imaging software (NIH ImageJ).
  • Htt Huntingtin
  • mice will be performed in blind. Results will be expressed as Standard Error Mean (SEM). Level of significance will be set for p ⁇ 0.05. Results will be analyzed with ANOVA with post-hoc correction with drug or genotype as main effect.
  • Example 16 FURTHER SCREENING OF HAT ACTIVATORS TO EXAMINE IF THEY AMELIORATE MOTOR ACTIVITY ABNORMALITIES IN MOUSE MODELS FOR PARKINSON'S DISEASE
  • PD is a degenerative disease with a neuronal death up to 75-95% of the dopamine neurons in the substantia nigra nucleus.
  • PD Parkinson's Disease
  • mice and WT treated with HAT activators PD mice and WT treated with vehicle.
  • mice will be sacrificed and their brains used for aggregated alpha-synuclein protein measurement.
  • HAT activation we will measure hippocampal acetyl-H4 levels.
  • Histone acetylation assay Western blot will be performed from snap-frozen in liquid nitrogen hippocampi. Tissue will be homogenized in RIPA buffer, then sonicated before centrifugation at 10,000 rpm for 5 min. Whole cell extracts will be electrophoresed on 10-20% gradient PAGE gel (Invitrogen) and then immunoblotted. Antibodies will be used at a 1:1,000 concentration for immunoblotting. All anti-histone antibodies will be purchased from Millipore. Immunoblot data will be quantified by measuring the band intensity using imaging software (NIH ImageJ).
  • alpha-synuclein levels will be performed on homogenates of frozen hemi-brains using an ⁇ -Synuclein ELISA Kit (Catalog # NS400; Millipore, Billerica, MA) according to manufacturer's instructions or via standard neuropathological methods (brain tissue histology).
  • mice will be performed in blind. Results will be expressed as Standard Error Mean (SEM). Level of significance will be set for p ⁇ 0.05. Results will be analyzed with ANOVA with post-hoc correction with drug or genotype as main effect.

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Claims (12)

  1. Composé, dans lequel le composé est :
    Figure imgb0036
     ou
    Figure imgb0037
  2. Procédé pour le criblage de composés selon la revendication 1 pour traiter des affections associées à des dépôts accumulés de peptide bêta-amyloïde, le procédé comprenant :
    a) l'administration d'un composé activateur de HAT selon la revendication 1 à un modèle animal d'accumulation de dépôts de peptide bêta-amyloïde ; et
    b) la sélection d'un composé activateur de HAT selon la revendication 1 qui peut moduler l'acétylation d'histones après administration du composé activateur de HAT dans un modèle animal d'accumulation de dépôts de peptide bêta-amyloïde.
  3. Procédé pour l'identification d'un composé activateur d'histone acétyltransférase (HAT) selon la revendication 1 pour traiter des affections associées à des dépôts accumulés de peptide bêta-amyloïde, dans lequel le procédé comprend la sélection d'un composé activateur de HAT selon la revendication 1 présentant une ou plusieurs des caractéristiques suivantes :
    a. la CE50 du composé est inférieure ou égale à environ 1 000 nM ;
    b. l'activité d'acétylation d'histones in vitro cible la protéine d'histone H2, H3, et/ou H4 ; et
    c. le composé passe à travers la barrière hématoencéphalique ; ou une combinaison de celles-ci.
  4. Procédé selon la revendication 3, dans lequel le composé présente une masse moléculaire inférieure à environ 500 Da, présente une surface polaire inférieure à environ 90 Â2, présente moins de 8 liaisons hydrogène, ou une combinaison de cela, afin de passer à travers la barrière hématoencéphalique.
  5. Composé selon la revendication 1 destiné à être utilisé en réduction de dépôts de protéine bêta-amyloïde (Aβ) chez un sujet par administration au sujet d'une quantité efficace d'une composition comprenant un composé activateur de HAT selon la revendication 1, ce qui diminue ainsi les dépôts de protéine Aβ chez le sujet.
  6. Composé selon la revendication 5 destiné à être utilisé, dans lequel le sujet présente des taux anormalement élevés de plaques de bêta-amyloïde ou
    dans lequel le sujet est atteint de maladie d'Alzheimer, de démence à corps de Lewy, de myosite à corps d'inclusion ou d'angiopathie amyloïde cérébrale, ou
    dans lequel le dépôt de protéine Aβ comprend un isomère Aβ40, un isomère Aβ42 ou une combinaison de ceux-ci.
  7. Composé selon la revendication 1 destiné à être utilisé en traitement de la maladie d'Alzheimer chez un sujet par administration à un sujet d'une quantité thérapeutique d'une composition pharmaceutique comprenant un composé selon la revendication 1, de préférence un composé activateur de HAT selon la revendication 1.
  8. Composé selon la revendication 1 destiné à être utilisé en augmentation de la fixation mnémonique chez un sujet atteint d'une maladie neurodégénérative par administration à un sujet d'une quantité thérapeutique d'une composition pharmaceutique comprenant le composé selon la revendication 1, ou
    composé selon la revendication 1 destiné à être utilisé en augmentation de la plasticité synaptique chez un sujet atteint d'une maladie neurodégénérative par administration à un sujet d'une quantité thérapeutique d'une composition qui augmente l'acétylation d'histones chez le sujet, dans lequel la composition comprend le composé selon la revendication 1, ou
    composé selon la revendication 1 destiné à être utilisé en traitement d'une maladie neurodégénérative chez un sujet par administration à un sujet d'une quantité thérapeutique d'une composition pharmaceutique comprenant le composé selon la revendication 1, ce qui traite ainsi la maladie neurodégénérative chez le sujet, ou
    composé selon la revendication 1 destiné à être utilisé en diminution de corps d'inclusion chez un sujet atteint d'un trouble neurodégénératif par administration au sujet d'une quantité efficace d'une composition comprenant un composé activateur de HAT selon la revendication 1, ce qui diminue ainsi les corps d'inclusion chez le sujet, ou
    composé selon la revendication 1 destiné à être utilisé en traitement d'un cancer chez un sujet par administration à un sujet d'une quantité thérapeutique d'une composition pharmaceutique comprenant le composé selon la revendication 1, ou
    composé selon la revendication 1 destiné à être utilisé en amélioration de symptômes de la maladie de Parkinson chez un sujet par administration à un sujet d'une quantité thérapeutique d'une composition pharmaceutique comprenant le composé selon la revendication 1 ; ou
    composé selon la revendication 1 destiné à être utilisé en amélioration de symptômes de la maladie de Parkinson par administration à un sujet d'une quantité thérapeutique d'une composition pharmaceutique comprenant un composé activateur de Hat selon la revendication 1, ou
    composé selon la revendication 1 destiné à être utilisé en traitement de la maladie de Huntington chez un sujet par administration à un sujet d'une quantité thérapeutique d'une composition pharmaceutique comprenant un composé selon la revendication 1 ; ou
    composé selon la revendication 1 destiné à être utilisé en traitement de la maladie de Huntington par administration à un sujet d'une quantité thérapeutique d'une composition pharmaceutique comprenant un composé activateur de HAT selon la revendication 1.
  9. Composé selon la revendication 1 destiné à être utilisé selon la revendication 5, 7, ou 8,
    dans lequel la quantité efficace est d'au moins environ 1 mg/kg de poids corporel, d'au moins environ 2 mg/kg de poids corporel, d'au moins environ 3 mg/kg de poids corporel, d'au moins environ 4 mg/kg de poids corporel, d'au moins environ 5 mg/kg de poids corporel, d'au moins environ 6 mg/kg de poids corporel, d'au moins environ 7 mg/kg de poids corporel, d'au moins environ 8 mg/kg de poids corporel, d'au moins environ 9 mg/kg de poids corporel, d'au moins environ 10 mg/kg de poids corporel, d'au moins environ 15 mg/kg de poids corporel, d'au moins environ 20 mg/kg de poids corporel, d'au moins environ 25 mg/kg de poids corporel, d'au moins environ 30 mg/kg de poids corporel, d'au moins environ 40 mg/kg de poids corporel, d'au moins environ 50 mg/kg de poids corporel, d'au moins environ 75 mg/kg de poids corporel, ou d'au moins environ 100 mg/kg de poids corporel, ou
    dans lequel la composition traverse la barrière hématoencéphalique, ou
    dans lequel la maladie neurodégénérative comprend l'adrénoleucodystrophie (ALD), l'alcoolisme, la maladie d'Alexander, la maladie d'Alper, la maladie d'Alzheimer, la sclérose latérale amyotrophique (la maladie de Lou Gehrig), l'ataxie télangiectasie, la maladie de Batten (également appelée maladie de Spielmeyer-Vogt-Sjögren-Batten), l'encéphalopathie spongiforme bovine (ESB), la maladie de Canavan, le syndrome de Cockayne, la dégénérescence cortico-basale, la maladie de Creutzfeldt-Jakob, l'insomnie fatale familiale, la dégénérescence lobaire frontotemporale, la maladie de Huntington, la démence associée au VIH, la maladie de Kennedy, la maladie de Krabbe, la démence à corps de Lewy, la neuroborréliose, la maladie de Machado-Joseph (l'ataxie spinocérébelleuse de type 3), l'atrophie multisystématisée, la sclérose en plaques, la narcolepsie, la maladie de Niemann-Pick, la maladie de Parkinson, la maladie de Pelizaeus-Merzbacher, la maladie de Pick, la sclérose latérale primitive, les maladies à prion, la paralysie supranucléaire progressive, le syndrome de Rett, la démence frontotemporale tau-positive, la démence frontotemporale tau-négative, la maladie de Refsum, la maladie de Sandhoff, la maladie de Schilder, la dégénérescence subaiguë combinée de la moelle épinière consécutive à une anémie pernicieuse, la maladie de Spielmeyer-Vogt-Sjögren-Batten, la maladie de Batten, l'ataxie spinocérébelleuse, l'amyotrophie spinale, la maladie de Steele-Richardson-Olszewski, le tabes dorsalis ou l'encéphalopathie toxique, ou
    dans lequel la plasticité synaptique comprend l'apprentissage, la mémoire ou une combinaison de ceux-ci, ou
    dans lequel la plasticité synaptique comprend la potentialisation à long terme (PLT), ou
    dans lequel le cancer comprend un lymphome B, un cancer du côlon, un cancer des poumons, un cancer du rein, un cancer de la vessie, un lymphome T, un myélome, une leucémie, une leucémie myéloïde chronique, une leucémie myéloïde aiguë, une leucémie lymphoïde chronique, une leucémie lymphoïde aiguë, une néoplasie hématopoïétique, un thymome, un lymphome, un sarcome, un cancer des poumons, un cancer du foie, un lymphome non hodgkinien, un lymphome hodgkinien, un cancer de l'utérus, un carcinome à cellules rénales, un hépatome, un adénocarcinome, un cancer du sein, un cancer pancréatique, un cancer du foie, un cancer de la prostate, un carcinome de la tête et du cou, un carcinome thyroïdien, un sarcome des tissus mous, un cancer des ovaires, un mélanome primaire ou métastatique, un carcinome squameux, un carcinome basocellulaire, un cancer du cerveau, un angiosarcome, un hémangiosarcome, un ostéosarcome, un fibrosarcome, un myxosarcome, un liposarcome, un chondrosarcome, un sarcome ostéogénique, un chordome, un angiosarcome, un endothéliosarcome, un lymphangiosarcome, un lymphangioendothéliosarcome, un synoviome, un cancer des testicules, un cancer du col de l'utérus, un cancer gastrointestinal, un mésothéliome, une tumeur d'Ewing, un léiomyosarcome, un rhabdomyosarcome, un cancer du côlon, un adénocarcinome, un carcinome des glandes sudoripares, un carcinome des glandes sébacées, un carcinome papillaire, une macroglobulinémie de Waldenström, un adénocarcinome papillaire, un cystadénocarcinome, un cancer bronchique, un carcinome de la voie biliaire, un choriocarcinome, un séminome, un carcinome embryonnaire, une tumeur de Wilms, un carcinome pulmonaire, un carcinome épithéliale, un cancer du col de l'utérus, une tumeur des testicules, un gliome, un astrocytome, un médulloblastome, un craniopharyngiome, un épendymome, un pinéalome, un hémangioblastome, un neurinome de l'acoustique, un oligodendrogliome, un méningiome, un rétinoblastome, une leucémie, un mélanome, un neuroblastome, un carcinome pulmonaire à petites cellules, un carcinome de la vessie, un lymphome, un myélome multiple, ou un carcinome médullaire.
  10. Composé selon la revendication 1 destiné à être utilisé selon la revendication 5, 7, ou 8, dans lequel le composé augmente l'acétylation d'histones.
  11. Composé selon la revendication 1 destiné à être utilisé selon la revendication 10, dans lequel l'acétylation d'histones comprend l'acétylation des histones H2B, H3, H4, ou d'une combinaison de celles-ci, ou
    dans lequel l'acétylation d'histones comprend l'acétylation de résidus de lysine d'histones H3K4, H3K9, H3K14, H4K5, H4K8, H4K12, H4K16 ou d'une combinaison de ceux-ci, ou
    dans lequel les corps d'inclusion comprennent des peptides bêta-amyloïdes, des protéines Tau natives et phosphorylées, l'alpha-synucléine native et phosphorylée, la lipofuscine, la TARDBP coupée (TDB-43) ou une combinaison de ceux-ci.
  12. Composé selon la revendication 1 destiné à être utilisé selon la revendication 8, dans lequel les symptômes de la maladie de Parkinson comprennent des tremblements, la bradykinésie, la dyskinésie, la rigidité, l'instabilité posturale, la dystonie, l'akathisie, la démence, la mauvaise coordination motrice globale ou une combinaison de ceux-ci, éventuellement
    dans lequel l'instabilité posturale comprend un mauvais équilibre, une mauvaise coordination ou une combinaison de ceux-ci.
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EP2509590B1 (fr) 2019-10-30
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JP2020059733A (ja) 2020-04-16
JP2016196459A (ja) 2016-11-24
JP2018027960A (ja) 2018-02-22
EP2509590A1 (fr) 2012-10-17
EP2509590A4 (fr) 2013-05-29
EP3632901A1 (fr) 2020-04-08
ES2764999T3 (es) 2020-06-05
JP6093180B2 (ja) 2017-03-08
ES2907862T3 (es) 2022-04-26

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